Phillip M. Friden

Anticandida Activity Is Retained in P-113, a 12-Amino-Acid Fragment of Histatin 5

ABSTRACT Through the analysis of a series of 25 peptides composed of various portions of the histatin 5 sequence, we have identified P-113, a 12-amino-acid fragment of histatin 5, as the smallest fragment that retains anticandidal activity comparable to that of the parent compound. Amidation of the P-113 C terminus increased the anticandidal activity of P-113 approximately twofold. The three histidine residues could be exchanged for three hydrophobic residues, with the fragment retaining anticandidal activity. However, the change of two or more of the five basic (lysine and arginine) residues to uncharged residues resulted in a substantial loss of anticandidal activity. A syntheticd-amino-acid analogue, P-113D, was as active againstCandida albicans as the l-amino-acid form. In vitro MIC tests in low-ionic-strength medium showed that P-113 has potent activity against Candida albicans, Candida glabrata, Candida parapsilosis, and Candida tropicalis. These results identify P-113 as a potential antimicrobial agent in the treatment of oral candidiasis.

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.

Photoactive Porphyrin Derivative with Broad-Spectrum Activity against Oral Pathogens In Vitro

ABSTRACT 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 nucleatumsubsp. 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.

P-113d, an Antimicrobial Peptide Active against Pseudomonas aeruginosa, Retains Activity in the Presence of Sputum from Cystic Fibrosis Patients

ABSTRACT 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 patients 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.

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.

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.

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.

Utilization of an endogenous cellular transport system for the delivery of therapeutics across the blood–brain barrier

Abstract The delivery of non-lipophilic compounds to the brain is severely limited by the presence of the blood–brain barrier (BBB). This barrier exists at the level of the capillary endothelial cells which form the vascular network within the brain. However, brain capillary endothelial cells do express specific receptors on their surface which are essential for the transport of nutrients and signaling molecules from the bloodstream to the brain. These endogenous transport pathways are potential portals for the transport of therapeutic molecules to the brain. The transferrin receptor is expressed at unusually high levels on the surface of brain capillary endothelial cells and is believed to be the primary, if not only, means for the transport of iron into the brain. It has been shown that antibodies that bind to the transferrin receptor selectively target BBB endothelium due to the high levels of this receptor expressed by these cells. These antibodies are then transported across the capillary endothelial cells into the brain and can function as carriers for the delivery of therapeutic peptides and proteins to the CNS. In particular, this drug delivery system has been used to transport nerve growth factor (NGF) across the BBB in a biologically active form and at levels sufficient to prevent the degeneration of NGF-dependent neurons.

Transport of Proteins Across the Blood-Brain Barrier via the Transferrin Receptor

Unlike most other organs in the body, the brain is separated from the blood by a protective cellular barrier known as the blood-brain barrier (BBB). The BBB, although essential in maintaining a defined biochemical environment within the brain, represents a formidable obstacle to the effective delivery of neuropharmaceutical agents from the bloodstream. The capillaries that supply blood to the tissues of the brain constitute this barrier (1,2). Brain capillary endothelial cells are joined together by tight intercellular junctions that form a continuous wall against the passive movement of substances from the blood to the brain. Also characteristic of these cells is a paucity of pinocytic vesicles, which limits the amount of non-selective fluid-phase transport across the capillary wall. Together, these features limit the penetration of blood-borne hydrophilic molecules into brain tissue.