P-113D, an antimicrobial peptide active against Pseudomonas aeruginosa, retains activity in the presence of sputum from cystic fibrosis patients.

Antimicrobial peptides are a source of novel agents that could be useful for treatment of the chronic lung infections that afflict cystic fibrosis (CF) patients. Efficacy depends on antimicrobial activity against the major pathogens of CF patients, Pseudomonas aeruginosa, Staphylococcus aureus, and Haemophilus influenzae, in the environment of the CF patient's airway. We describe the in vitro efficacies of derivatives of histatins, which are histidine-rich peptides produced by the salivary glands of humans and higher primates. P-113, a peptide containing 12 of the 24 amino acid residues of the parent molecule, histatin 5, retained full antibacterial activity and had a good spectrum of activity in vitro against the prominent pathogens of CF patients. However, P-113 was not active in the presence of purulent sputum from CF patients. In contrast, P-113D, the mirror-image peptide with the amino acid residues in the D configuration, was stable in sputum, was as active as P-113 against pathogens of CF patients in the absence of sputum and retained significant activity in the presence of sputum from CF patients. Recombinant human DNase, which effectively liquefies sputum, enhanced the activity of P-113D in undiluted sputum against both exogenous (added) bacteria and endogenous bacteria. Because of its properties, P-113D shows potential as an inhalant in chronic suppressive therapy for CF patients.

Novel periodontal drug delivery system for treatment of periodontitis.

A conceptually novel periodontal drug delivery system (DDS) is described that is intended for treatment of microbial infections associated with periodontitis. The DDS is a composite wafer with surface layers possessing adhesive properties, while the bulk layer consists of antimicrobial agents, biodegradable polymers, and matrix polymers. The wafers contain poly(lactic-co-glycolic acid) as the main bioerodible component used in the bulk layer and ethyl cellulose applied as a matrix polymer enabling diffusion-controlled release. Starch and other polymers in combination with AgNO(3) serve as coatings adhesive to the teeth. In vitro experiments demonstrate that the wafers are capable of zero-order release of antimicrobial agents such as silver nitrate, benzylpenicillin, and tetracycline, for over 4 weeks.

Photoactive porphyrin derivative with broad-spectrum activity against oral pathogens In vitro.

Photodynamic therapy (PDT) has historically been used as a means to treat cancerous tumors but has recently been used to kill bacterial cells through the use of targeted photosensitizers. PDT is a potential adjunct to scaling and root planing in the treatment of periodontal disease. However, the effectiveness of porphyrin derivatives against microorganisms has been limited because some gram-negative bacteria are refractory to photodynamic treatment with these agents. We have designed a porphyrin derivative conjugated to a pentalysine moeity that endows the molecule with activity against gram-positive and gram-negative bacteria. Whereas the porphyrin, chlorin e6, showed in vitro activity against a limited spectrum of bacteria, chlorin e6 conjugated to pentalysine showed in vitro activity against all oral microorganisms tested, including Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, Bacteroides forsythus, Campylobacter rectus, Eikenella corrodens, Fusobacterium nucleatum subsp. polymorphum, Actinomyces viscosus, and the streptococci. Potent antimicrobial activity (>/=5-log-unit reduction in the numbers of CFU per milliliter) was retained in the presence of up to 25% whole sheep blood. The use of potent, selective agents such as this chlorin e6-pentalysine conjugate to more effectively reduce the pathogenic bacteria in the periodontal pocket may be a significant tool for the treatment of periodontal disease.

Sustained release of silver from periodontal wafers for treatment of periodontitis.

Periodontal wafers intended to treat the underlying infections in patients with periodontitis have been developed. The wafers consist of poly(lactic-co-glycolic acid) as a primary bioerodible polymeric component, poly(ethylene glycol) as a plasticizer and encapsulation aid, and silver nitrate as the antimicrobial agent. The wafers are capable of sustained in vitro release of bioactive silver for at least 4 weeks. The wafers exhibit silver release that follows erosion kinetics, confirming a bulk erosion/release mechanism. In clinical evaluation, sustained release of silver at bactericidal levels for at least 21 days is observed. Staining of hard and soft tissues due to the released silver is minimal and reversible.

Inhibition of experimental gingivitis in beagle dogs with topical salivary histatins.

Histatins, histidine-rich proteins found within parotid and submandibular secretions, are a novel class of endogenous peptides with antimicrobial properties. This masked, randomized, placebo-controlled preclinical investigation examined the effect of 3 topical histatins on the development of plaque and gingivitis in beagle dogs. 16, female, 1-year-old beagles were brought to optimal gingival health by mechanical scaling and polishing followed by rigorous daily tooth brushing. At the conclusion of this pretreatment period, dogs were randomly divided into 4 groups for the application of test formulations, and were placed on a plaque-promoting diet. Test agents included 3 synthetic salivary histatins (histatin 5, P-113 and P-113D) which were incorporated in hydroxypropyl methylcellulose gel at a concentration of 0.125%, and a placebo, or negative control, which was the gel vehicle alone. Throughout the 10-week treatment period, test formulations (2.0 ml) were applied 2 x daily to all premolar teeth using a Monojet syringe. Plaque formation and gingival inflammation were assessed using the plaque (PI) and gingival (GI) indices on days 0, 7, 14, 21, 28, 42, 56 and 70. Furthermore, bleeding to probing was recorded as a percent of sites (%BOP) and according to the modified sulcus bleeding index (mSBI). Comparisons among groups and between group pairs (active versus placebo) were made with Kruskal-Wallis tests with the average of data over the interval, days 14-42, being the primary focus of the analysis. From baseline to day 7, all groups expressed similar indices. Thereafter, overall significant differences among the groups were noted at day 42 for PI, at days 21, 28, 42 and 70 for GI, and at days 14 and 28 for %BOP (p < 0.05). In particular, beagles treated with P-113 demonstrated significantly lower PI scores at day 42 (p < 0.05), significantly lower GI scores from days 21 through 42 (p < 0.05), and significantly lower %BOP scores at days 14 and 28 (p < 0.05) compared to beagles treated with placebo. Beagles treated with P-113D exhibited significantly lower GI at day 42 compared to the placebo (p < 0.05). For the primary analysis conducted over the midtreatment interval (days 14-42), significant differences were detected for all parameters except mSBI (p < 0.05). Accordingly, significantly lower PI scores were found for P-113, lower GI scores for P-113 and P-113D, and lower %BOP for P-113 and P-113D compared to placebo (p < 0.05). These data indicate that in the beagle model, salivary histatins, P-113 and P-113D, topically applied, can significantly reduce clinical signs of plaque formation and gingival inflammation.

Transcytosis of protein through the mammalian cerebral epithelium and endothelium. III. Receptor-mediated transcytosis through the blood-brain barrier of blood-borne transferrin and antibody against the transferrin receptor.

Diferric-transferrin (Tf; 80K mol. wt.) and the OX26 antibody (150K mol. wt.) against the transferrin receptor (TfR) were evaluated in the rat at light and ultrastructural levels as potential vehicles for the blood to brain transcellular transfer (transcytosis) of native horseradish peroxidase (40K mol. wt.), which by itself does not cross the blood-brain barrier (BBB). OX26, the Fab fragment of OX26 (50K mol. wt.), and Tf complexed to two ferric ions were conjugated to HRP irreversibly in a 1:1 molar ratio. The indirect immunoperoxidase technique with OX26 as the monoclonal primary antibody applied to the surface of cryostat sections or delivered intravenously to the live rat revealed TfRs on BBB capillaries, arterioles, and venules; TfRs were absent on non-BBB vessels supplying the circumventricular organs (i.e., median eminence, choroid plexus). OX26-HRP and OX26(Fab)-HRP delivered intravenously and diferric-Tf-HRP administered into the carotid artery labeled BBB vessels throughout the CNS without discernible disruption of the BBB or extravasation of the blood-borne probes into the brain parenchyma. No reaction product for the probes was observed in sites deficient in a BBB. Each of the macromolecular conjugates was endocytosed by BBB endothelia and labeled presumptive endocytic vesicles, endosomes, and dense bodies. OX26-HRP and Tf-HRP, but not OX26(Fab)-HRP, appeared to undergo transcytosis through BBB endothelia for subsequent labeling of perivascular cells. Distinct differences in the intracellular and extracellular distributions between OX26-HRP and Tf-HRP were identified: (1) endocytosis and sequestration of blood-borne OX26-HRP within BBB endothelia were more prominent than those for diferric-Tf-HRP; (2) only OX26-HRP labeled the Golgi complex in BBB endothelia; (3) peroxidase labeling of CNS perivascular clefts and perivascular cells in rats receiving diferric-Tf-HRP was conspicuous at less than 1 h postinjection but not so in rats with blood-borne OX26-HRP at 5 min through 6 h postinjection; and (4) peroxidase-labeled CNS neurons and glial cells were identified readily in rats receiving diferric-Tf-HRP. The results suggest that the receptor-mediated, transendothelial transfer of Tf-HRP from blood to brain is more efficient and direct than that of OX26-HRP. Labeling of the Golgi complex in BBB endothelia with blood-borne OX26-HRP implies that the transendothelial transfer of OX26-HRP follows intraendothelial pathways associated with the process of adsorptive transcytosis. A diagram is provided depicting the possible intracellular and transcellular pathways within BBB endothelia available to blood-borne diferric-Tf and OX26 as vectors for delivery into the CNS of non-lipid-soluble macromolecules that otherwise are denied entry by the blood-brain fluid barriers.

Characterization, receptor mapping and blood-brain barrier transcytosis of antibodies to the human transferrin receptor.

Monoclonal antibodies to the human transferrin receptor were screened for binding to capillary vessels in human, monkey, rabbit and rat brain tissue. Two antibodies were selected that bind both human and monkey but not rabbit or rat microvessels. With recombinant fragments of the human receptor, both antibodies were shown to bind to a region of the extracellular portion of the receptor that is relatively variable among species. Binding, which was characterized by using purified receptor and K562 cells, was not reduced by excess transferrin, indicating that the antibodies bind the receptor at a site different from that of transferrin. When the antibodies were radiolabeled and injected i.v. into cynomolgous monkeys, they distributed selectively to brain but not to other organs or tissues. The antibodies were found almost exclusively in the brain parenchyma, rather than the capillaries, indicating that they had transcytosed the blood-brain barrier. These results show that antibodies to the human transferrin receptor cross the blood-brain barrier and may be useful for noninvasive delivery of therapeutic proteins to the central nervous system.

Atrophy of cholinergic basal forebrain neurons following excitotoxic cortical lesions is reversed by intravenous administration of an NGF conjugate.

Nerve growth factor (NGF) has been shown to sustain the viability and modulate the function of cholinergic basal forebrain neurons. However, under normal circumstances, NGF does not cross the blood-brain barrier (BBB) following systemic administration making this neurotrophin unavailable to NGF-responsive neurons within the central nervous system (CNS). Recently, a non-invasive method for delivering NGF to the brain was established in which NGF was conjugated to an antibody directed against the transferrin receptor (OX-26) [15, 16]. This conjugation facilitates the transfer of NGF from the systemic circulation to the CNS via the transferrin transport system. In the present study, we tested whether intravenous administration of an OX-26-NGF conjugate could reverse the atrophy of cholinergic basal forebrain neurons following removal of the target sites. Lesions of the left cerebral cortex were created by epidural application of N-methyl-D-aspartic acid (NMDA). Seventy-five days later, cholinergic nucleus basalis neurons were atrophic ipsilateral to the lesion relative to the contralateral side in control rats receiving intravenous injections of vehicle or a non-conjugated mixture of OX-26 and NGF. In contrast, intravenous injections of the OX-26-NGF conjugate restored the size of nucleus basalis perikarya to within normal limits relative to the unlesioned contralateral side. Immunohistochemical studies using rat serum albumen antisera indicated that the BBB was closed at the time of treatment indicating that this trophic effect did not result from NGF crossing through a compromised BBB at the site of the lesion. These data demonstrate that systemic administration of a neurotrophic factor-antibody conjugate, intended to circumvent the BBB, can provide trophic influences to degenerating cholinergic basal forebrain neurons. These data support the emerging concept that the conjugate method can facilitate the transfer of impermeable therapeutic compounds across the BBB.

Enhanced uptake of rsCD4 across the rodent and primate blood-brain barrier after conjugation to anti-transferrin receptor antibodies.

The delivery to the brain of nonlipophilic therapeutic compounds, especially proteins, is severely hindered by the presence of the blood-brain barrier, which is formed by the tightly apposed brain capillary endothelial cells. However, brain endothelial cells do possess specific receptor-mediated transport mechanisms so that substances required by the brain can cross the blood-brain barrier. By use of monoclonal antibodies that bind to the transferrin receptor present on the luminal surface of brain capillary endothelial cells, we have taken advantage of the transport system responsible for the delivery of iron to the brain to deliver recombinant human soluble CD4 (rsCD4), a potential anti-HIV therapeutic, across the blood-brain barrier. Anti-transferrin receptor antibody-rsCD4 conjugates were synthesized with a disulfide linkage and characterized in vitro. Experiments that use immunohistochemistry to localize these conjugates after intravenous administration into the tail vein of rats have shown that both the carrier antibody and the protein "passenger" accumulate in brain capillaries. The carrier-mediated delivery of radiolabeled protein across the blood-brain barrier in vivo was also examined in both rodents and primates. With use of the technique of capillary depletion in rats, the amount of rsCD4 in the capillary fraction of the brain, which reaches a maximal value within 1 hr postinjection, was shown to decrease with time, whereas the amount in the brain parenchyma increased, which suggests that the protein was delivered across the blood-brain barrier. In primates rsCD4 levels in the brain were increased 5-fold when the protein was administrated intravenously in the form of an anti-transferrin receptor antibody-rsCD4 conjugate.

Pathway across blood-brain barrier opened by the bradykinin agonist, RMP-7.

The route taken by lanthanum (MW 139) across cerebral endothelium was delineated when the blood-brain barrier was opened by RMP-7, a novel bradykinin agonist. Balb C mice were infused through a jugular vein with LaCl3 with or without RMP-7 (5 micrograms/kg). Ten minutes later, the brains were fixed with aldehydes and processed for electron microscopy. The patency of the junctions between endothelial cells was estimated by counting the number of junctions penetrated by LaCl3. Tracer penetrated the junctions in about 25% of microvessels in vehicle infused, control mice and about 58% in the RMP-7 group, where more junctions per vessel were also penetrated. The LaCl3 then penetrated the basal lamina in about 20% of all microvessels in the RMP-7 group, versus 0.50% in the control group. From the basal lamina, the tracer entered perivascular spaces in about 13% of all microvessels in the RMP-7 group and about 0.07% in the controls. Very few endocytic pits or vesicles in the RMP-7 group were labeled, so LaCl3 did not cross endothelium by transcytosis. The increased number of tight junctions penetrated by tracer and its spread into periendothelial basal lamina and interstitial clefts indicated, therefore, a paracellular route of exudation in the RMP-7 treated animals.

Effects of transferrin receptor antibody-NGF conjugate on young and aged septal transplants in oculo.

The purpose of this study was to investigate the effects of nerve growth factor (NGF) conjugated to a monoclonal transferrin receptor antibody (OX-26) on septal transplants in oculo. Three different doses of OX-26-NGF conjugate (0.3, 3, and 50 micrograms/injection) were injected into the tail vein of young adult hosts 2, 4, and 6 weeks following intraocular transplantation of fetal forebrain tissue containing septal nuclei. Intravenous injections of OX-26 alone, NGF alone, and saline served as controls. An increase in intraocular tissue growth, as well as an increase in the intensity of immunoreactivity for p75 receptors and acetylcholinesterase, was observed following peripheral OX-26-NGF administration at the two highest doses tested. In addition, aged host rats with 16-month-old intraocular septal grafts were injected intravenously with OX-26 or OX-26-NGF (10 micrograms NGF/injection) every 2 weeks until the transplants were 24 months old. The intensity of choline acetyltransferase-like (ChAT) staining appeared to be greater and the cell bodies were larger with more processes in aged transplants in hosts treated with the OX-26-NGF conjugate than in aged OX-26-treated subjects. The present results suggest that peripheral OX-26-NGF can deliver biologically active NGF across the blood-brain barrier and have dose-dependent positive effects on both aged and developing cholinergic neurons in septal transplants.

Intravenous administration of a transferrin receptor antibody-nerve growth factor conjugate prevents the degeneration of cholinergic striatal neurons in a model of Huntington disease.

Intrastriatal injections of quinolinic acid induce a pattern of neuronal degeneration similar to that seen in Huntington disease. In the present study, nerve growth factor (NGF) crossed the blood-brain barrier in a dose-dependent fashion following intravenous infusion when conjugated to an antibody directed against the transferrin receptor (OX-26). Intravenous injections of the OX-26-NGF conjugate selectively prevented the loss of striatal choline acetyltransferase-immunoreactive neurons which normally occurs following quinolinic acid administration relative to control rats receiving vehicle or a nonconjugated mixture of OX-26 and NGF. These data demonstrate that a neurotrophic factor-antibody conjugate can prevent the degeneration of central NGF-responsive neurons following systemic administration.

Receptor-mediated transport of therapeutics across the blood-brain barrier.

The delivery of nonlipophilic compounds to the brain is severely hindered by the blood-brain barrier (BBB). However, brain capillary endothelial cells, which form the BBB, do possess specific receptor-mediated transport mechanisms that potentially can be exploited as a means to transport therapeutic molecules to the brain. We have found that antibodies that bind to the transferrin receptor selectively target BBB endothelium, are transported into the brain, and can function as carriers for the delivery of compounds, including proteins, to the central nervous system. This drug delivery system has been used to transport nerve growth factor across the BBB in a biologically active form and at levels sufficient to prevent the degeneration of nerve growth factor-dependent neurons.

Synthesis, in vitro kinetics, and in vivo studies on protein conjugates of AZT: evaluation as a transport system to increase brain delivery.

Our method to increase the delivery of polar drugs to the central nervous system is via drug-protein conjugates with proteins that interact with and cross brain capillary endothelial cells. As a model for drugs containing a reactive hydroxyl group, AZT was conjugated via a succinate linker to two such protein carriers, the highly cationic histone H1 and an anti-transferrin receptor antibody, OX-26. The protein carriers were selected on the basis of their ability to interact with brain capillary endothelial cells by absorptive or receptor-mediated events, respectively. An in vitro pH profile of the rate of AZT release indicated that the observed hydrolysis proceeds by a specific base-catalysis mechanism. At 37 degrees C, the release of AZT proceeded at a rate approximately 10-fold faster (Kobs approximately 8 x 10(-4) min-1) than expected for a simple ester (AZT succinate; Kobs approximately 1.25 x 10(-4) min-1). Using simple model systems, product analysis revealed that intramolecular cyclization of the succinate linker accounts for the observed rate enhancement. Drug delivery in vivo was assessed using immunohistochemical techniques and quantitative brain uptake measurements with singly and doubly labeled AZT-OX-26 conjugates. Immunohistochemical staining of brain sections showed the colocalization of AZT and OX-26 in the brain vasculature. Therefore, drug can be linked to the antibody without affecting the targeting property of the antibody. Furthermore, an in vivo time course using radiolabeled conjugate showed that AZT is delivered to the brain capillaries but is not transported into the brain parenchyma with the antibody.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood-brain barrier penetration and in vivo activity of an NGF conjugate.

Nerve growth factor (NGF) is essential for the survival of both peripheral ganglion cells and central cholinergic neurons of the basal forebrain. The accelerated loss of central cholinergic neurons during Alzheimer's disease may be a determinant of dementia in these patients and may therefore suggest a therapeutic role for NGF. However, NGF does not significantly penetrate the blood-brain barrier, which makes its clinical utility dependent on invasive neurosurgical procedures. When conjugated to an antibody to the transferrin receptor, however, NGF crossed the blood-brain barrier after peripheral injection. This conjugated NGF increased the survival of both cholinergic and noncholinergic neurons of the medial septal nucleus that had been transplanted into the anterior chamber of the rat eye. This approach may prove useful for the treatment of Alzheimer's disease and other neurological disorders that are amenable to treatment by proteins that do not readily cross the blood-brain barrier.

Selective transport of an anti-transferrin receptor antibody through the blood-brain barrier in vivo.

The brain capillary endothelium, which makes up the blood-brain barrier (BBB) in vivo, expresses high concentrations of transferrin receptor, and recent studies show that an antitransferrin receptor monoclonal antibody may function as a BBB drug transport vector. The present report examines the pharmacokinetics of clearance of radiolabeled antitransferrin receptor monoclonal antibody from the bloodstream in rats in vivo, and also assesses the extent to which brain selectively extracts the antibody from the blood compared to other peripheral organs such as liver, kidney, myocardium, or lung. [125I]Mouse immunoglobulin G2a control antibody was cleared monoexponentially with a half-time of 9.8 +/- 2.3 h. The clearance of the [3H]OX-26 antitransferrin receptor antibody from blood was biexponential with half-times of 2.2 +/- 0.8 min (61 +/- 10% of clearance) and 3.9 +/- 0.2 h (39 +/- 4% of clearance). The OX-26 antibody was rapidly taken up by liver during the first 60 min after injection, but this uptake reached rapid saturation, and hepatic OX-26 content actually declined subsequent to the first hour after injection. In contrast, brain continuously extracted the OX-26 antibody from the bloodstream, and the brain volume of distribution of OX-26 reached a value 18-fold greater than the volume of distribution of the mouse immunoglobulin G2a at 5 h after injection. There was no specific uptake of the OX-26 by myocardium or lung, and minor uptake by kidney was observed that also reached saturation within the first 60 min after injection.(ABSTRACT TRUNCATED AT 250 WORDS)

Anti-transferrin receptor antibody and antibody-drug conjugates cross the blood-brain barrier.

Delivery of nonlipophilic drugs to the brain is hindered by the tightly apposed capillary endothelial cells that make up the blood-brain barrier. We have examined the ability of a monoclonal antibody (OX-26), which recognizes the rat transferrin receptor, to function as a carrier for the delivery of drugs across the blood-brain barrier. This antibody, which was previously shown to bind preferentially to capillary endothelial cells in the brain after intravenous administration (Jefferies, W. A., Brandon, M. R., Hunt, S. V., Williams, A. F., Gatter, K. C. & Mason, D. Y. (1984) Nature (London) 312, 162-163), labels the entire cerebrovascular bed in a dose-dependent manner. The initially uniform labeling of brain capillaries becomes extremely punctate approximately 4 hr after injection, suggesting a time-dependent sequestering of the antibody. Capillary-depletion experiments, in which the brain is separated into capillary and parenchymal fractions, show a time-dependent migration of radiolabeled antibody from the capillaries into the brain parenchyma, which is consistent with the transcytosis of compounds across the blood-brain barrier. Antibody-methotrexate conjugates were tested in vivo to assess the carrier ability of this antibody. Immunohistochemical staining for either component of an OX-26-methotrexate conjugate revealed patterns of cerebrovascular labeling identical to those observed with the unaltered antibody. Accumulation of radiolabeled methotrexate in the brain parenchyma is greatly enhanced when the drug is conjugated to OX-26.