Mode-selective inhibitory effects of eugenol on the mouse TRPV1 channel.

The transient receptor potential vanilloid 1 (TRPV1) channel is a polymodal receptor in sensory nerves and involved in pain sensation. TRPV1 has at least three distinct activation modes that are selectively induced by different stimuli capsaicin, noxious heat, and protons. Although many mode-selective TRPV1 antagonists have been developed for their anticipated analgesic effects, there have been few successful reports because of adverse effects due to burn injuries and hyperthermia. Eugenol is a vanilloid that has been used as an analgesic in the dental treatment, and its TRPV1 activation ability has been reported. However, our knowledge about the underlying mechanisms of the antagonistic effects of eugenol on TRPV1 activation induced by three different modes is limited. Here, we show that eugenol dose-dependently inhibited the capsaicin-activated inward currents of mouse TRPV1 expressed in human embryonic kidney 293 (HEK293) cells. Under low pH conditions, low concentrations of eugenol only enhanced the proton-induced TRPV1 currents, whereas high eugenol concentrations initially potentiated but then immediately abrogated TRPV1 currents. Finally, eugenol had no modulatory effects on heat-activated TRPV1 in electrophysiological and Fura-2-based Ca2+ imaging experiments. Our results demonstrate that eugenol is a mode-selective antagonist of TRPV1 and can be evaluated as a lead compound of analgesics targeting TRPV1 without serious side effects.

Calcium inhibits dihydropyridine-stimulated increases in opening and unitary conductance of a plant Ca²+ channel.

We have previously characterized the "RCA" channel (root Ca²+ channel), a voltage-dependent, Ca²+-permeable channel found in plasma membrane-enriched vesicles from wheat roots incorporated into artificial planar lipid bilayers. Earlier work indicated that this channel was insensitive to 1,4-dihydropyridines (DHPs, such as nifedipine and 202-791). However, the present study shows that this channel is sensitive to DHPs, but only with submillimolar Ca²+, when the probability of channel opening is reduced, with flickery closures becoming increasingly evident as Ca²+ activity decreases. Under these ionic conditions, addition of nanomolar concentrations of (+) 202-791 or nifedipine caused an increase in both the probability of channel opening and the unitary conductance. It is proposed that there is a competitive interaction between Ca²+ and DHPs at one of the Ca²+-binding sites involved in Ca²+ permeation and that binding of a DHP to one of the Ca²+-permeation sites facilitates movement of other calcium ions through the channel. The present study shows that higher plant Ca²+-permeable channels can be greatly affected by very low concentrations of DHPs and that channel sensitivity may vary with the ionic conditions of the experiment. The results also indicate interesting structural and functional differences between plant and animal Ca²+-permeable channels.

Inhibition of Alzheimer's amyloid toxicity with a tricyclic pyrone molecule in vitro and in vivo.

Small beta-amyloid (Abeta) 1-42 aggregates are toxic to neurons and may be the primary toxic species in Alzheimer's disease (AD). Methods to reduce the level of Abeta, prevent Abeta aggregation, and eliminate existing Abeta aggregates have been proposed for treatment of AD. A tricyclic pyrone named CP2 is found to prevent cell death associated with Abeta oligomers. We studied the possible mechanisms of neuroprotection by CP2. Surface plasmon resonance spectroscopy shows a direct binding of CP2 with Abeta42 oligomer. Circular dichroism spectroscopy reveals monomeric Abeta42 peptide remains as a random coil/alpha-helix structure in the presence of CP2 over 48 h. Atomic force microscopy studies show CP2 exhibits similar ability to inhibit Abeta42 aggregation as that of Congo red and curcumin. Atomic force microscopy closed-fluid cell study demonstrates that CP2 disaggregates Abeta42 oligomers and protofibrils. CP2 also blocks Abeta fibrillations using a protein quantification method. Treatment of 5x familial Alzheimer's disease mice, a robust Abeta42-producing animal model of AD, with a 2-week course of CP2 resulted in 40% and 50% decreases in non-fibrillar and fibrillar Abeta species, respectively. Our results suggest that CP2 might be beneficial to AD patients by preventing Abeta aggregation and disaggregating existing Abeta oligomers and protofibrils.

Arbaclofen placarbil, a novel R-baclofen prodrug: improved absorption, distribution, metabolism, and elimination properties compared with R-baclofen.

Baclofen is a racemic GABA(B) receptor agonist that has a number of significant pharmacokinetic limitations, including a narrow window of absorption in the upper small intestine and rapid clearance from the blood. Arbaclofen placarbil is a novel transported prodrug of the pharmacologically active R-isomer of baclofen designed to be absorbed throughout the intestine by both passive and active mechanisms via the monocarboxylate type 1 transporter. Arbaclofen placarbil is rapidly converted to R-baclofen in human and animal tissues in vitro. This conversion seems to be primarily catalyzed in human tissues by human carboxylesterase-2, a major carboxylesterase expressed at high levels in various tissues including human intestinal cells. Arbaclofen placarbil was efficiently absorbed and rapidly converted to R-baclofen after oral dosing in rats, dogs, and monkeys. Exposure to R-baclofen was proportional to arbaclofen placarbil dose, whereas exposure to intact prodrug was low. Arbaclofen placarbil demonstrated enhanced colonic absorption, i.e., 5-fold higher R-baclofen exposure in rats and 12-fold higher in monkeys compared with intracolonic administration of R-baclofen. Sustained release formulations of arbaclofen placarbil demonstrated sustained R-baclofen exposure in dogs with bioavailability up to 68%. In clinical use, arbaclofen placarbil may improve the treatment of patients with gastroesophageal reflux disease, spasticity, and numerous other conditions by prolonging exposure and decreasing the fluctuations in plasma levels of R-baclofen.

The binding of donepezil with external mouth of K+-channels of molluscan neurons.

Earlier, we have shown a strong inhibitory effect of donepezil on K+-current of molluscan neurons (Solntseva et al., Comp Biochem Physiol 144, 319-326, 2007). In the present work, a possible interaction of donepezil with the external mouth of the channel was examined using, as a tool, tetraethylammonium (TEA), a classical antagonist of potassium channels. Experiments were conducted in isolated neurons of snail Helix aspersa using the two-microelectrode voltage-clamp technique. A high-threshold slow-inactivating K+-current involving Ca2+-dependent (I (C)) and Ca2+-independent (I (K)) components was recorded. The I (C) was estimated at 30 mV, and I (K) at 100 mV. The IC(50) values for blocking effect of donepezil on I (C) varied from 5.0 to 8.9 microM in different cells. Corresponding values for I (K) varied from 4.9 to 9.9 microM. The IC(50) values for blocking effect of TEA on I (C) lied in the range of 200 to 910 microM, and on I (K) lied in the range of 100 to 990 microM. The comparison of the effects of donepezil and TEA on the same cells revealed significant correlation between IC(50) values of these effects. The value of Spearman coefficient of correlation (r) was 0.77 for I (C) (P < 0.05), and 0.82 for I (K) (P < 0.05). In the presence of TEA, the effect of donepezil, both on I (C) and I (K), appears significantly weaker than in control solution. Dose-response curves of donepezil effect both on I (C) and I (K) were shifted right along horizontal axis when donepezil was applied in combination with TEA. Results suggest that TEA interferes with donepezil and precludes the occupation by donepezil of its own site. We suppose that the site for donepezil is situated near the TEA site with possible overlap.

Treatment of hepatocellular carcinoma in mice with PE38KDEL type I mutant-loaded poly(lactic-co-glycolic acid) nanoparticles conjugated with humanized SM5-1 F(ab') fragments.

We reported previously the development of SMFv-PE38KDEL type I mutant (PE38KDEL-I; Mut-I), a recombinant immunotoxin in which a single-chain antibody derived from mouse SM5-1 monoclonal antibody is genetically fused to PE38KDEL-I. In comparison with the SMFv-PE38KDEL wild-type, Mut-I showed improved therapeutic efficacy and reduced toxicity. To overcome the problems associated with the immune response to the Pseudomonas exotoxin A (PE) component of Mut-I, we have constructed PE38KDEL-I-loaded poly(lactic-co-glycolic acid) nanoparticles conjugated with F(ab') fragments of a humanized SM5-1 monoclonal antibody (PE-NP-S). PE-NP-S specifically bound to SM5-1 binding protein-expressing hepatocellular carcinoma cell lines and was then internalized by these cells, resulting in significant cytotoxic effect. In SM5-1 binding protein-overexpressing tumor xenograft model, administration of PE-NP-S significantly inhibited tumor development and induced tumor regression. Moreover, PE-NP-S was shown to be much weaker in inducing vascular leakage syndrome in mice than Mut-I. The LD(50) of PE-NP-S was about 4-fold higher than that of Mut-I. Remarkably, PE-NP-S was of low immunogenicity in development of anti-PE neutralizing antibodies in vivo and was less susceptible to inactivation by anti-PE neutralizing antibodies compared with Mut-I. In conclusion, the resultant PE-NP-S possessed increased cancer therapeutic efficacy and had reduced nonspecific toxicity and immunogenicity, suggesting that it is a potential candidate in cancer therapy.

Dissolved organic matter and estrogenic potential of landfill leachate.

The estrogenic potentials of leachate samples collected at Laogang Sanitary Landfill in Shanghai, China were measured together with the associated dissolved organic matter (DOM) in leachate samples. Over 99% of the DOM in fresh leachate was removed upon 3-7 years of landfill, leaving only DOM with strong fluorescent activity. Anoxic or aerobic treatment of landfill leachate can further degrade DOM of MW<300 Da and transform those with fluorescent activity of MW>10(5) Da to those of 2000-10(5) Da. Neither landfilling nor storage in anoxic pond effectively removed estrogenic potential of leachate. Fractionation test revealed that residual organic matters of MW 3000-14000 Da and of <600 Da with high UV254 contributed most of the estrogenic activities in leachate. Aerobic SBR treatment considerably reduced the estrogenic potential of these organic matters in leachate.

Promoter scanning for transcription inhibition with DNA-binding polyamides.

When targeted to sequences adjacent to a TATA element, pyrrole-imidazole (Py-Im) polyamides inhibit the DNA binding activity of TATA box binding protein (TBP) and basal transcription by RNA polymerase II. In the present study, we scanned the human immunodeficiency virus type 1 promoter for polyamide inhibition of TBP binding and transcription using a series of DNA constructs in which a polyamide binding site was placed at various distances from the TATA box. Polyamide interference with either TBP-DNA or TFIID-TFIIA-DNA contacts both upstream and downstream of the TATA element resulted in inhibition of transcription. Our results define important protein-DNA interactions outside of the TATA element and suggest that transcription inhibition of selected gene promoters can be achieved with polyamides that target unique sequences within these promoters at a distance from the TATA element. Our studies also demonstrate the utility of the Py-Im polyamides for discovery of functionally important protein-DNA contacts involved in transcription.

Biophysical and biological studies of some polymer grafted metallo-intercalators.

Two water-soluble polymer-copper(II) complexes, [Cu(ip)2(BPEI)](ClO4)2·H2O (Complex 1) and [Cu(dppz)2BPEI](ClO4)2·H2O (Complex 2) with different degree of coordination have been synthesized and characterized. The interaction between the prepared complexes and CTDNA has been assessed by various physico-chemical methods The spectroscopic and the cyclic voltammetry studies have revealed that both the complexes interact with CTDNA through intercalation binding mode. Among the two complexes, Complex 2 has higher binding affinity with CTDNA. The antiproliferative activity of the complexes has been examined on human breast cancer cells, MDAMB231, adopting various techniques. The results indicate that both the polymer-copper(II) complexes are effective against the breast cancer cell line and the order of the activity is consistent with the DNA-binding ability.

In vitro and in vivo characterization of F-97013-GD, a partial 5-HT1A agonist with antipsychotic- and antiparkinsonian-like properties.

In order to better define the role of 5-HT(1A) receptors in the modulation of extrapyramidal motor functions, we investigated the effect of 5-HT(1A) agonists on tacrine-induced tremulous jaw movements (TJM) in rats, a putative model of parkinsonian tremor. Acute injection of 5-HT(1A) agonists 8-OH-DPAT and buspirone dose-dependently counteracted the tacrine-induced oral movements (ED(50)=0.04 and 1.0mg/kg, respectively), an effect reversed by the selective 5-HT(1A) antagonist WAY 100,635. In contrast to classical antipsychotics, the atypical antipsychotics risperidone (ED(50)=0.3mg/kg) and clozapine (ED(50)=1.5mg/kg) blocked the oral movements induced by the cholinomimetic agent at or below the doses required for suppression of conditioned avoidance response. The compound F-97013-GD (6-methyl-2-[4-(naphtylpiperazin-1-yl)butyl]-3-(2H)-pyridazinone), a putative antipsychotic drug that in functional in vitro and in vivo assays behaved as a mixed dopamine D(2)-antagonist and 5-HT(1A)-partial agonist, also displayed a potent antitremorgenic effect in this paradigm (ED(50)=0.5mg/kg). Interestingly, pretreatment with WAY 100,635 blocked the inhibitory effect of F-97013-GD but not that of clozapine. The 5-HT depleting agent para-chlorophenylalanine (PCPA) partially attenuated tacrine-induced TJM but did not block the suppressive effect of 5-HT(1A) agonists. In addition, only high doses of F-97013-GD induced catalepsy in rodents and, like 8-OH-DPAT and clozapine, the compound reversed the haloperidol-induced catalepsy in rats. These results show that 5-HT(1A) receptors play a role in the regulation of tacrine-induced TJM and suggest that their activation by novel antipsychotics may not only reduce the extrapyramidal side effects EPS liability, but also be effective in the treatment of parkinsonian tremor.

Regulation of ASAP1 by phospholipids is dependent on the interface between the PH and Arf GAP domains.

ASAP1 is an Arf GAP with a PH domain immediately N-terminal to the catalytic Arf GAP domain. PH domains are thought to regulate enzymes by binding to specific phosphoinositide lipids in membranes, thereby recruiting the enzyme to a site of action. Here, we have examined the functional relationship between the PH and Arf GAP domains. We found that GAP activity requires the cognate PH domain of ASAP1, leading us to hypothesize that the Arf GAP and PH domains directly interact to form the substrate binding site. This hypothesis was supported by the combined results of protection and hydrodynamic studies. We then examined the role of the PH domain in the regulation of Arf GAP activity. The results of saturation kinetics, limited proteolysis, FRET and fluorescence spectrometry support a model in which regulation of the GAP activity of ASAP1 involves a conformational change coincident with recruitment to a membrane surface, and a second conformational change following the specific binding of phosphatidylinositol 4,5-bisphosphate.

Prevention of the interaction between HVEM, herpes virus entry mediator, and gD, HSV envelope protein, by a Keggin polyoxotungstate, PM-19.

One of the Keggin-type heteropolyoxotungstates (K7[PTi2W10O40]6H2O:PM-19) is a potent inhibitor of the replication of herpes simplex virus (HSV) both standard strain 169 and the thymidine kinase-defective strain YS-4C-1 in vitro and in vivo. HSV envelope protein, gD, is necessary for virus entry into the cells. Some cellular molecules, such as HVEM, were reported to act as cofactors during the viral entry step. We determined whether PM-19 prevents these interactions between HSV-gD and HVEM. These activities were investigated using the Ciphergen and BIACORE system. Using a protein chip, many kinds of gD-specific binding proteins were captured, but these proteins could not be identified. Several proteins in these gD-binding proteins were inhibited its interaction with gD due to the presence of PM-19. Using the BIACORE system, the affinity of PM-19 to gD was low, because PM-19 has no direct inactivation activity against the virion. The specific binding of HVEM to the gD was shown as KD of 1.1e-9. The affinity of PM-19 for HVEM was high (KD:2e-9). To determine the competitive binding, the PM-19 (10 microg/ml) and several concentrations of HVEM solution mixtures were injected over the gD-fixed sensor surface. Each binding signal was stable in the range of approximately 270-300 RU. In the case of the addition of PM-19 to HVEM solution, the binding signals were elevated by PM-19 dose dependently. These results suggest that the bindings of PM-19 to gD are not disturbed by the presence of HVEM. PM-19 prevents the interaction between HVEM and gD.

Administration of a decoy against the activator protein-1 binding site suppresses neointimal thickening in rabbit balloon-injured arteries.

BACKGROUND: Transcription factor activator protein-1 (AP-1) is activated and upregulated in injured arterial smooth muscle cells in vivo, yet the exact role of the AP-1--related pathway in vascular disease in vivo has remained unclear. We examined the role of the transfer of synthetic double-stranded cis-element decoy oligodeoxynucleotides (ODNs) in balloon-injured rabbit carotid arteries and the effects of these ODNs on neointimal thickening. METHODS AND RESULTS: Transfection of fluorescein isothiocyanate--labeled ODNs using the hemagglutinating virus of Japan liposome method resulted in widespread distribution of fluorescent nuclear signals over the entire medial layer in injured arteries. Gel mobility shift assay revealed that AP-1 DNA binding was activated and that the AP-1 decoy reduced AP-1 DNA binding activity as a result of specific binding affinity to AP-1 in vivo. In morphometric analyses, AP-1 decoy led to a significant reduction in the neointimal area and a significant reduction in cell number and transforming growth factor-beta(1) production of human aortic smooth muscle cells under conditions of platelet-derived growth factor stimulation. CONCLUSIONS: Because AP-1 decoy transfection in vivo dramatically prevented neointimal thickening in balloon-injured arteries, AP-1 may be a useful molecular target for gene therapy to reduce restenosis.

Amino-terminal dimerization of an erythropoietin mimetic peptide results in increased erythropoietic activity.

BACKGROUND: Erythropoietin (EPO), the hormone involved in red blood cell production, activates its receptor by binding to the receptor's extracellular domain and presumably dimerizing two receptor monomers to initiate signal transduction. EPO-mimetic peptides, such as EMP1, also bind and activate the receptor by dimerization. These mimetic peptides are not as potent as EPO, however. The crystal structure of the EPO receptor (EBP) bound to EMP1 reveals the formation of a complex consisting of two peptides bound to two receptors, so we sought to improve the biological activity of EPO-mimetic peptides by constructing covalent dimers of EMP1 and other peptide mimetics linked by polyethylene glycol (PEG). RESULTS: The potency of the PEG-dimerized EPO peptide mimetics both in vitro and in vivo was improved up to 1,000-fold compared to the corresponding peptide monomers. The dimers were constructed using peptide monomers which have only one reactive amine per molecule, allowing us to conclude that the increase in potency can be attributed to a structure in which two peptides are linked through their respective amino termini to the difunctional PEG molecule. In addition, an inactive peptide was converted into a weak agonist by PEG-induced dimerization. CONCLUSIONS: The potency of previously isolated peptides that are modest agonists of the EPO receptor was dramatically increased by PEG-induced dimerization. The EPO receptor is thought to be dimerized during activation, so our results are consistent with the proposed 2:2 receptor : peptide stoichiometry. The conversion of an inactive peptide into an agonist further supports the idea that dimerization can mediate receptor activation.

Targeted therapy of colorectal neoplasia with rapamycin in peptide-labeled pegylated octadecyl lithocholate micelles.

Many powerful drugs have limited clinical utility because of poor water solubility and high systemic toxicity. Here, we formulated a targeted nanomedicine, rapamycin encapsulated in pegylated octadecyl lithocholate micelles labeled with a new ligand for colorectal neoplasia, LTTHYKL peptide. CPC;Apc mice that spontaneously develop colonic adenomas were treated with free rapamycin, plain rapamycin micelles, and peptide-labeled rapamycin micelles via intraperitoneal injection for 35days. Endoscopy was performed to monitor adenoma regression in vivo. We observed complete adenoma regression at the end of therapy. The mean regression rate for peptide-labeled rapamycin micelles was significantly greater than that for plain rapamycin micelles, P<0.01. On immunohistochemistry, we observed a significant reduction in phospho-S6 but not ß-catenin expression and reduced tumor cell proliferation, suggesting greater inhibition of downstream mTOR signaling. We observed significantly reduced renal toxicity for peptide-labeled rapamycin micelles compared to that of free drug, and no other toxicities were found on chemistries. Together, this unique targeted micelle represents a potential therapeutic for colorectal neoplasia with comparable therapeutic efficacy to rapamycin free drug and significantly less systemic toxicity.

Binding and cytotoxic trafficking of cholesterol hydroperoxides by sterol carrier protein-2.

Redox-active cholesterol hydroperoxides (ChOOHs) generated by oxidative stress in eukaryotic cells may propagate cytotoxic membrane damage by undergoing one-electron reduction or, at low levels, act as mobile signaling molecules like H2O2. We discovered that ChOOHs can spontaneously translocate between membranes or membranes and lipoproteins in model systems, and that this can be accelerated by sterol carrier protein-2 (SCP-2), a nonspecific lipid trafficking protein. We found that cells overexpressing SCP-2 were more susceptible to damage/toxicity by 7α-OOH (a free radical-generated ChOOH) than control cells, and that this correlated with 7α-OOH delivery to mitochondria. The methods used for obtaining these results and for establishing that cellular SCP-2 binds and traffics 7α-OOH are described in this chapter.

Liposome-mediated peptide loading of MHC-DR molecules in vivo.

Amino acid residues 3-15 of mycobacterial HSP60 define a dominant T-cell epitope for HLA-DR3+ve humans and Mamu-DR3+ve rhesus monkeys. Our results show that Mamu-DR3 molecules on PBMC can be efficiently loaded in vivo with the above-mentioned peptides when they are intravenously injected encapsulated in liposomes, but not in the free form. Mamu-DR3 loading is abolished by encapsulation of a nonstimulatory peptide. These results have implications for the delivery of therapeutic peptides in vivo.

Assessment of an automated solid phase competitive fluoroimmunoassay for benzoylecgonine in untreated urine.

A new solid phase fluoroimmunoassay using a fully automated flow fluorometer adapted for urinalysis of drug metabolites is described. Fluorescein-conjugated benzoylecgonine (FL-BE) and monoclonal antibodies (mAb) against benzoylecgonine (BE) were the reagents used for demonstration. The solid phase consisted of anti-BE mAbs immobilized on the surface of polymethyl methacrylate (PMMA) beads. Free BE in solution competed with FL-BE and reduced bead-bound fluorescence in a concentration-dependent manner. The binding of FL-BE to the anti-BE mAb reached steady-state within minutes. FL-BE was not bound by uncoated beads nor beads coated with non-specific proteins or IgG. The signal-to-noise ratio was 33, and the sensitivity of the assay was 2 ng ml(-1) for BE. The effective concentration of BE was 1 to 100 ng ml(-1), with an IC50 value of 12 ng ml(-1). The mAb showed equal affinities for BE, cocaine, and cocaethylene, but a five order-of-magnitude lower affinity for ecgonine and ecgonine methylester. In a double-blind comparison using clinical urine samples, the data from this single-step competitive assay had excellent agreement with results obtained using a fiber-optic biosensor (FOB), and the EMIT assay performed commercially. The assay provided kinetic data rapidly and can be used to detect small analytes for which antibodies and fluorescein conjugates are available. The affinity of the mAb for FL-BE, calculated from kinetic analysis of the time course of the on and off reaction, was 2.25 x 10(-9) M.

Competitive and glycine/NMDA receptor antagonists attenuate withdrawal-induced behaviours and increased hippocampal acetylcholine efflux in morphine-dependent rats.

The present study has examined the glycine/N-methyl-D-aspartate (NMDA) receptor antagonist, R-(+)-3-amino-1-hydroxypyrrolid-2-one (R-(+)-HA-966) and the competitive NMDA receptor antagonist, cis-4-(phosphonomethyl)piperidine-2-carboxylic acid (CGS 19755) on the behavioural syndrome and increased hipppocampal acetylcholine efflux induced during morphine-withdrawal in the rat. Subcutaneous naltrexone (1 mg/kg) injection, 48 hr after implantation of a 75 mg morphine pellet, induced a robust withdrawal syndrome consisting of wet dog shakes, ejaculations, mouth movement, ptosis, irritability to touch and diarrhoea. Pretreatment with the alpha2-adrenoceptor agonist, clonidine (0.1-0.4 mg/kg), R-(+)-HA-966 (10-60 mg/kg) or CGS 19755 (5 or 10 mg/kg) significantly reduced the incidence of withdrawal behaviours. In addition, all three compounds significantly attenuated the increase in hippocampal acetylcholine efflux induced following naltrexone (1 mg/kg, s.c.) injection in morphine-dependent rats. These results provide further evidence demonstrating that NMDA receptor antagonists attenuate both the behavioural and neurochemical effects observed during morphine withdrawal in the rat.

Conjugation of acetylcholine receptor-protecting Fab fragments with polyethylene glycol results in a prolonged half-life in the circulation and reduced immunogenicity.

Antibodies to the acetylcholine receptor (AChR) cause AChR loss, resulting in the disease, myasthenia gravis (MG). The majority of the pathogenic antibodies seem to be directed against the main immunogenic region (MIR) of the AChR. In contrast to the intact antibodies, Fab fragments of anti-AChR antibodies are not themselves pathogenic and such fragments of anti-MIR monoclonal antibodies (mAbs) protect the AChR in vitro and in vivo against the pathogenic antibodies. However, Fab fragments have a very short in vivo half-life and are immunogenic, obstacles which must be overcome before their clinical use can be envisaged. We investigated the effect of conjugating Fab fragments to polyethylene glycol (PEG), a method known to increase the in vivo half-life and reduce the immunogenicity of proteins. When the Fab' fragments of two rat anti-MIR mAbs (nos. 35 and 195) were conjugated to methoxy-PEG-maleimide, the conjugates retained about 10% of their AChR binding activity and efficiently protected the AChR against the binding and modulating activity of myasthenic antibodies. Their in vivo half-life in rats was approximately 15 times longer than that of the unconjugated Fab' fragment and they were much less immunogenic in mice. This work represents an important step towards the clinical use of AChR-protective anti-MIR Fabs, but further improvements are needed before their clinical use is attempted.