Paul Kiptoo

Vaccine-like Controlled-Release Delivery of an Immunomodulating Peptide to Treat Experimental Autoimmune Encephalomyelitis

The objective of this work is to use colloidal gel from alginate-chitosan-PLGA complex to deliver Ac-PLP-BPI-NH₂-2 peptide in a controlled-release manner as a vaccine-like therapeutic to suppress experimental autoimmune encephalomyelitis (EAE) in the mouse model. Oppositely charged PLGA nanoparticles were prepared by a solvent diffusion method. The carboxyl group of the alginate and the amine group of the chitosan coated the nanoparticles with negative and positive charges, respectively. The peptide (Ac-PLP-BPI-NH₂-2), designed to bind to MHC-II and ICAM-1 simultaneously, was formulated into the colloidal gel by physical mixture. Vaccine-like administration of the peptide-loaded colloidal gel (Ac-PLP-BPI-NH₂-2-NP) was achieved by subcutaneous (sc) injection to EAE mice. Disease severity was measured using clinical scoring and percent change in body weight. Cytokine production was determined using the splenocytes from Ac-PLP-BPI-NH₂-2-NP-treated mice and compared to that of controls. Ac-PLP-BPI-NH₂-2-NP suppressed and delayed the onset of EAE as well as Ac-PLP-BPI-NH₂-2 when delivered in a vaccine-like manner. IL-6 and IL-17 levels were significantly lower in the Ac-PLP-BPI-NH₂-2-NP-treated mice compared to the mouse group treated with blank colloidal gel, suggesting that the mechanism of suppression of EAE is due to a shift in the immune response away from Th17 production. The results of this study suggest that a one-time sc administration of Ac-PLP-BPI-NH₂-2 formulated in a colloidal gel can produce long-term suppression of EAE by reducing Th17 proliferation.

Enhancement of Drug Absorption through the Blood Brain Barrier and Inhibition of Intercellular Tight Junction Resealing by E-cadherin Peptides

E-cadherin-mediated cell-cell interactions in the zonula adherens play an important role in the formation of the intercellular tight junctions found in the blood-brain barrier. However, it is also responsible for the low permeation of drugs into the brain. In this study, HAV6 peptide derived from the EC1 domain of E-cadherin was found to enhance the permeation of ¹⁴C-mannitol and [³H(G)]-daunomycin through the blood-brain barrier of the in situ rat brain perfusion model. In addition, HAV6 peptide and verapamil have a synergistic effect in enhancing the BBB permeation of daunomycin. A new intercellular-junction resealing assay was also developed using Caco-2 monolayers to evaluate new peptides (BLG2, BLG3, and BLG4) derived from the bulge regions of the EC2, EC3, and EC4 domains of E-cadherin. BLG2 and BLG4 peptides but not BLG3 peptides were found to be effective in blocking the resealing of the intercellular junctions. The positive control peptides (ADT10, ADT6, and HAV10) block the resealing of the intercellular junctions in a concentration-dependent manner. All these findings suggest that E-cadherin-derived peptides can block E-cadherin-mediated cell-cell interactions. These findings demonstrate that cadherin peptides may offer a useful targeted permeation enhancement of therapeutic agents such as anticancer drugs into the brain.

Antigen‐specific blocking of CD4‐Specific immunological synapse formation using BPI and current therapies for autoimmune diseases

In this review, we discuss T‐cell activation, etiology, and the current therapies of autoimmune diseases (i.e., MS, T1D, and RA). T‐cells are activated upon interaction with antigen‐presenting cells (APC) followed by a “bulls eye”‐like formation of the immunological synapse (IS) at the T‐cell–APC interface. Although the various disease‐modifying therapies developed so far have been shown to modulate the IS and thus help in the management of these diseases, they are also known to present some undesirable side effects. In this study, we describe a novel and selective way to suppress autoimmunity by using a bifunctional peptide inhibitor (BPI). BPI uses an intercellular adhesion molecule‐1 (ICAM‐1)‐binding peptide to target antigenic peptides (e.g., proteolipid peptide, glutamic acid decarboxylase, and type II collagen) to the APC and therefore modulate the immune response. The central hypothesis is that BPI blocks the IS formation by simultaneously binding to major histocompatibility complex‐II and ICAM‐1 on the APC and selectively alters the activation of T cells from TH1 to Treg and/or TH2 phenotypes, leading to tolerance.

Antigen-Specific Suppression of Experimental Autoimmune Encephalomyelitis by a Novel Bifunctional Peptide Inhibitor: Structure Optimization and Pharmacokinetics

The objective of this study was to optimize the in vivo activity of proteolipid protein (PLP)-bifunctional peptide inhibitor (BPI) molecule to suppress experimental autoimmune encephalomyelitis (EAE) in SJL/J mice and evaluate pharmacokinetic profiles of PLP-BPI. PLP-BPI is constructed via conjugation of myelin PLP139-151 with CD11a237-246-derived peptide (LABL) via a spacer. The hypothesis is that PLP-BPI binds simultaneously to major histocompatibility complex-II and intercellular adhesion molecule-1 on the antigen-presenting cell (APC) and inhibits the formation of the immunological synapse during T-cell and APC interactions. In this study, the structure of BPI was modified by varying the spacer and was evaluated in the EAE model. Intravenous injections of BPI derivatives inhibited the onset, severity, and incidence of EAE more effectively and induced a lower incidence of anaphylaxis than that produced by unmodified PLP-BPI. As anticipated, production of interleukin-17, a proinflammatory cytokine commonly found in elevated levels among multiple sclerosis (MS) patients, was significantly lower in Ac-PLP-BPI-PEG6- or Ac-PLP-BPI-NH2-2-treated mice than in phosphate-buffered saline-treated mice. These results suggest that BPI-type molecules can be modified to achieve more efficient and better tolerated BPI-based derivatives for the treatment of MS.

Prophylactic and Therapeutic Suppression of Experimental Autoimmune Encephalomyelitis by a Novel Bifunctional Peptide Inhibitor

The objective was to optimize and evaluate the in vivo activities of our novel bifunctional peptide inhibitor (BPI), which alters immune response in autoimmune diseases by modulating the immunological synapse formation. Previously, we have designed PLP-BPI and GAD-BPI by conjugating myelin proteolipid protein (PLP)(139-151) and glutamic acid decarboxylase (GAD)(208-217), respectively, with CD11a(237-246) via a spacer peptide. PLP-BPI and GAD-BPI suppressed the disease progression in experimental autoimmune encephalomyelitis (EAE) and in type-1 diabetes, respectively. In this study, various PLP-BPI derivatives were synthesized and evaluated in the EAE model. Intravenous injections of PLP-BPI derivatives prevented the disease progression more efficiently than did unmodified PLP-BPI. Production of IL-17, a potent proinflammatory cytokine found commonly among MS patients, was significantly low in Ac-PLP-BPI-NH(2)-2-treated mice. Treatment given after the disease onset could dramatically ameliorate the disease. BPI induced anaphylactic responses at a lower incidence than PLP(139-151). In conclusion, PLP-BPI derivatives can effectively suppress the disease severity and morbidity of EAE by post-onset therapeutic treatment as well as prophylactic use.

Immune response to controlled release of immunomodulating peptides in a murine experimental autoimmune encephalomyelitis (EAE) model

The effects of controlled release on immune response to an immunomodulating peptide were evaluated in a murine experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis (MS). The peptide, Ac-PLP-BPI-NH(2)-2 (Ac-HSLGKWLGHPDKF-(AcpGAcpGAcp)(2)-ITDGEATDSG-NH(2); Ac = acetyl, Acp = epsilon aminocaproic acid) was designed to suppress T-cell activation in response to PLP(139-151), an antigenic peptide in MS. Poly-lactide-co-glycolide (PLGA) microparticles containing Ac-PLP-BPI-NH(2)-2 (8+/-4 microm, 1.4+/-0.2% (w/w)) were prepared by a powder-in oil-in water emulsion-solvent evaporation method, sterilized and administered subcutaneously (s.c.) to SJL/J (H-2(s)) mice in which EAE had been induced by immunization with PLP(139-151). Treatment groups received Ac-PLP-BPI-NH(2)-2: (i) in solution by repeated i.v. or s.c. injection, (ii) in solution co-administered with blank PLGA microparticles, (iii) in solution co-administered with Ac-PLP-BPI-NH(2)-2 loaded microparticles, and (iv) as Ac-PLP-BPI-NH(2)-2 loaded microparticles. Administration of Ac-PLP-BPI-NH(2)-2 as an s.c. solution produced clinical scores and maintenance of body weight comparable to i.v. solution, but with reduced overall survival, presumably due to anaphylaxis. Administration as s.c. microparticles provided a somewhat less effective reduction in symptoms but with no toxicity during treatment. Thus, the results suggest that s.c. administration of Ac-PLP-BPI-NH(2)-2 microparticles can provide pharmacological efficacy and reduction in dosing frequency without increased toxicity.

Pathways and progress in improving drug delivery through the intestinal mucosa and blood-brain barriers.

One of the major hurdles in developing therapeutic agents is the difficulty in delivering drugs through the intestinal mucosa and blood-brain barriers (BBB). The goal here is to describe the general structures of the biological barriers and the strategies to enhance drug delivery across these barriers. Prodrug methods used to improve drug penetration via the transcellular pathway have been successfully developed, and some prodrugs have been used to treat patients. The use of transporters to improve absorption of some drugs (e.g., antiviral agents) has also been successful in treating patients. Other methods, including blocking the efflux pumps to improve transcellular delivery, and modulation of cell-cell adhesion in the intercellular junctions to improve paracellular delivery across biological barriers, are still in the investigational stage.

Modulation of blood-brain barrier permeability in mice using synthetic E-cadherin peptide.

The present work characterizes the effects of synthetic E-cadherin peptide (HAV) on blood–brain barrier (BBB) integrity using various techniques including magnetic resonance imaging (MRI) and near-infrared fluorescent imaging (NIRF). The permeability of small molecular weight permeability marker gadolinium diethylenetriaminepentaacetate (Gd-DTPA) contrast agent, the large molecular weight permeability marker, IRDye 800CW PEG, and the P-glycoprotein (P-gp) efflux transporter contrast agent, rhodamine 800 (R800), were examined in the presence and absence of HAV peptide. The results consistently demonstrated that systemic iv administration of HAV peptide resulted in a reversible disruption of BBB integrity and enhanced the accumulation of all the dyes examined. The magnitude of increase ranged from 2-fold to 5-fold depending on the size and the properties of the permeability markers. The time frame for BBB disruption with HAV peptide was rapid, occurring within 3–6 min following injection of the peptide. Furthermore, modulation of BBB permeability was reversible with the barrier integrity being restored within 60 min of the injection. The increased BBB permeability observed following HAV peptide administration was not attributable to changes in cerebral blood flow. These studies support the potential use of cadherin peptides to rapidly and reversibly modulate BBB permeability of a variety of therapeutic agents.

Modulation of Intercellular Junctions by Cyclic-ADT Peptides as a Method to Reversibly Increase Blood–Brain Barrier Permeability

It is challenging to deliver molecules to the brain for diagnosis and treatment of brain diseases. This is primarily because of the presence of the blood-brain barrier (BBB), which restricts the entry of many molecules into the brain. In this study, cyclic-ADT peptides (ADTC1, ADTC5, and ADTC6) have been shown to modify the BBB to enhance the delivery of marker molecules [e.g., (14) C-mannitol, gadolinium-diethylenetriaminepentacetate (Gd-DTPA)] to the brain via the paracellular pathways of the BBB. The hypothesis is that these peptides modulate cadherin interactions in the adherens junctions of the vascular endothelial cells forming the BBB to increase paracellular drug permeation. In vitro studies indicated that ADTC5 had the best profile to inhibit adherens junction resealing in Madin-Darby canine kidney cell monolayers in a concentration-dependent manner (IC50 = 0.3 mM) with a maximal response at 0.4 mM. Under the current experimental conditions, ADTC5 improved the delivery of (14) C-mannitol to the brain about twofold compared with the negative control in the in situ rat brain perfusion model. Furthermore, ADTC5 peptide increased in vivo delivery of Gd-DTPA to the brain of Balb/c mice when administered intravenously. In conclusion, ADTC5 has the potential to improve delivery of diagnostic and therapeutic agents to the brain.

Immune Tolerance Induction against Experimental Autoimmune Encephalomyelitis (EAE) Using A New PLP-B7AP Conjugate that Simultaneously Targets B7/CD28 Costimulatory Signal and TCR/MHC-II Signal

Most of the current therapies used in the treatment of multiple sclerosis (MS) are either ineffective or have adverse side effects. As such, there is a need to develop better therapies that specifically target myelin-specific aberrant immune cells involved in CNS inflammation without compromising the general immune system. In the present study, we developed a new bifunctional peptide inhibitor (BPI) that is effective and specific. Our BPI (PLP-B7AP) is composed of an antigenic peptide from myelin proteolipid protein (PLP139–151) and a B7 antisense peptide (B7AP) derived from CD28 receptor. The main hypothesis is that PLP-B7AP simultaneously targets MHC-II and B7-costimulatory molecules on the surface of antigen presenting cells (APC) and possibly alters the differentiation of naïve T cells from inflammatory to regulatory phenotypes. Results showed that PLP-B7AP was very effective in suppressing experimental autoimmune encephalomyelitis (EAE) compared to various controls in a mouse model. PLP-B7AP was effective when administered both before and after disease induction. Secreted cytokines from splenocytes isolated during periods of high disease severity and remission indicated that PLP-B7AP treatment induced an increased production of anti-inflammatory cytokines and inhibited the production of pro-inflammatory cytokines. Further, analysis of cortical brain tissue sections showed that PLP-B7AP treated mice had significantly lower demyelination compared to the control group. All these taken together indicate that the T cell receptor (TCR) and the CD28 receptor can be targeted simultaneously to improve efficacy and specificity of potential MS therapeutics.