WO1991016064A1 - Procedes et compositions permettant de rendre permeable, de maniere reversible, la barriere hemato-encephalique - Google Patents

Procedes et compositions permettant de rendre permeable, de maniere reversible, la barriere hemato-encephalique Download PDF

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Publication number
WO1991016064A1
WO1991016064A1 PCT/US1991/002533 US9102533W WO9116064A1 WO 1991016064 A1 WO1991016064 A1 WO 1991016064A1 US 9102533 W US9102533 W US 9102533W WO 9116064 A1 WO9116064 A1 WO 9116064A1
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WO
WIPO (PCT)
Prior art keywords
cell wall
bbb
csf
brain
eubacteria
Prior art date
Application number
PCT/US1991/002533
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English (en)
Inventor
Alexander Tomasz
Elaine Tuomanen
Original Assignee
The Rockefeller University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The Rockefeller University filed Critical The Rockefeller University
Priority to JP91508041A priority Critical patent/JPH05506660A/ja
Publication of WO1991016064A1 publication Critical patent/WO1991016064A1/fr

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/742—Spore-forming bacteria, e.g. Bacillus coagulans, Bacillus subtilis, clostridium or Lactobacillus sporogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5005—Wall or coating material
    • A61K9/5063—Compounds of unknown constitution, e.g. material from plants or animals
    • A61K9/5068—Cell membranes or bacterial membranes enclosing drugs
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • This invention relates to permeabilization of the blood brain barrier (BBB) . It is particularly related to a method of enhancing the permeability of BBB.
  • the BBB in order to permit passage of chemotherapeutic agents, diagnostic agents, imaging agents, drugs and the like useful in the treatment of brain disorders or diseases, from the intravascular compartment to the interstitial fluid
  • CSF cerebrospinal fluid
  • the present invention relates to the use of the cell wall of the bacterium Streptococcus pneumoniae and other eubacteria as well as fragments thereof (more fully defined below) 20 to reversibly permeabilize the BBB and allow passage of useful therapeutic agents across the BBB.
  • the BBB is a continuous boundary between the blood and both the interstitial fluid (IF) and the 25 CSF of the brain. It is composed of a layer of endothelial cells, the cerebral capillary endothelium, that serves as an effective barrier against the entry into the brain's tissue of serum components of both high and low molecular sizes.
  • the restriction against entry of such substances into the brain and the CSF is due to the unique structure of the cerebral capillary endothelium.
  • the BBB breaks down to its maximal known extent. Morphologically, one or both of two events occur: 1) tight junctions open between endothelial cells; b) an increased number of pinocytotic vesicles appear which may transport components across the BBB. Either one or both of these phenomena may be associated with enhanced movement of serum components into the CSF and the brain intersitital fluid; this and the accumulation of leukocytes in the CSF are the hallmarks of bacterial meningitis.
  • Goldstein and Betz in their aforementioned article briefly mention two modes of administering medicines to the brain.
  • the resistance of the BBB to penetration is lowered by the injection of a hyperosmotic sugar solution into the carotid artery.
  • the second mode which is still experimental, involves direct injection of the therapeutic agent into the CSF.
  • this latter method can easily lead to serious mechanical injury during introduction of the needle.
  • WCF cell wall fragments
  • the BBB is permeabilized, or its permeability is enhanced to allow a more effective passage of therapeutic agents into the brain tissues and/or the CSF.
  • the enhanced permeability of the BBB is accomplished by the introduction (via parenteral, preferably intravenous injection) of an effective dose of a purified cell wall or CWF.
  • the resulting temporary disruption of the BBB then permits the passage therethrough of a desired therapeutic agent.
  • the therapeutic agent is preferably administered after the CWF, although it may be administered before the CWF or simultaneously therewith. These alternative possibilities are encompassed herein by the term "together with”.
  • CWF including the whole cell wall from Streptococcus pneumoniae, but it is not so limited.
  • CWFs from other eubacteria are also useful. These include, for example Micrococcus lysodekticus, Bacillus subtilis, Escherichia coli and Staphylococcus aureus. Additionally, the invention is applicable to the use of products in which the CWF is covalently or otherwise bound to the material to be transported across the BBB.
  • the CWF may bind to the BBB, act as a receptor for the material to be transported and subsequently effect the transfer of the material across the BBB.
  • the single drawing herein shows the structures for the two major building blocks of the pneumonococcal cell wall. It should be understood, however, that the invention is not limited to these structures.
  • Streptococcus pneumoniae is a human pathogen which has been known for a long time.
  • the morphological, biochemical and physiological features of the cell wall of this bacterium have been described in several scientific and medical publications (see, e.g., the articles cited above).
  • Purified cell wall of Streptococcus pneumonia when introduced intrathecally, can open the BBB to the passage of cellular and serum soluble components of the blood, which are typical signs and symptoms of pneumococcal meningitis in animal models of this disease.
  • the same highly purified pneumococcal cell wall or CWFs were introduced parenterally, e.g. by intravenous (iv) administration into rabbits. It was found that such iv administration resulted in the opening of BBB as evidenced by the enhanced passage of otherwise poorly permeable substances (including some therapeutic agents) into the brain and CSF.
  • the pneumococcal cell wall is a complex macromolecule or polymer composed of two major polymers: a peptidoglycan and a ribitol-phosphate teichoic acid of unusually complex structure which contains phosphorylcholine.
  • the structural diversity within this macromolecule has recently been shown to be very large, particularly within the peptide side chains of the peptidoglycan network.
  • a number of chemically distinct degradation products may be generated from the intact cell wall by a variety of chemical or enzymatic techniques in vitro as well as in vivo under the influence of host enzymes or due to the triggering of the activity of wall degrading enzymes in the pneumococcus or other eubacteria during treatment with antibiotics.
  • the structures shown in the figure are not the only fragments which may be obtained from the cell wall. The method of isolation of several fragments is described in the reference 4. Those cell wall fragments and the cell wall itself are useful in the practice of this invention.
  • the CWFs used in the invention when introduced intrathecally, permit the passage of cellular and serum soluble components of the blood through the BBB, including for example albumin, serum protein and leucocytes. When injected parenterally the products do not appear to open the BBB to the passage of leucocytes.
  • the cell wall is a polymeric macromolecule composed of a wide variety of different molecules joined one to the other including glycopeptides, teichoic acid, lipids, glycans and others.
  • the term "cell wall fragments" includes any portion of this macromolecule which is capable of opening the BBB to passage by components of the blood when administered intravenously. It includes, therefore, the component polymers of the cell wall, the biosynthetic precursors of these polymers as well as their in vivo and in vitro fragmentation products whether produced by chemical or enzymatic means. It includes, also, synthetic analogs of these materials having similar physiological activity.
  • Streptococcus pneumoniae R 6 (a known unencapsulated strain originally derived from strain R36S an encapsulated type II strain available from the Laboratories of Rockefeller University) was grown in one-liter batches of a chemically defined medium (Cden) to a cell concentration of about 5 x 10 cfu/ml.
  • the cells were harvested by centrifugation, resuspended in 5 ml of saline and the resulting suspension was submerged in a boiling water bath for 15 minutes to inactivate the autolytic enzyme.
  • the suspension was then transferred to a cuvette of the Mickle disintegrator (Hampton, Middlesex, England) mixed with an equal volume of glass beads (Ballotini no. t 5 13; 100 urn diameter; 3M Company, St. Paul,
  • the cells, resuspended in 0.1 M Tris-HCl buffer (ph 8.0) containing ImM MgCl_ were treated for 37°C with pancreatic DNase I (50 ug/ml) plus RNase (100 ug/ml) for 2 hours followed by trypsin (100 ug/ml) plus 10 25 mM CaCl 2 for 10-12 hours.
  • pancreatic DNase I 50 ug/ml
  • RNase 100 ug/ml
  • trypsin 100 ug/ml
  • 10 25 mM CaCl 2 for 10-12 hours.
  • All enzyme preparations were of crystalline grade and were obtained from Worthington Bioche icals, Freehold, New Jersey.
  • Cell wall was sedimented by centrifugation (10,000 g for 30 minutes) and resuspended in ml of 2% SDS at 30 90-100°C in a water bath for 30 minutes.
  • the first is a high molecular weight glycan-teichoic acid complex containing all the cell wall teichoic acid and glycan with some of the stem peptides still attached
  • the second is a lower molecular weight mixture of most of the cell wall peptides.
  • the cell wall hydrolysates were centrifuged (15,000 g for 30 minutes) and the supernatants lyophilized and then dissolved in 1 ml of saline and layered on a G75 Sephadex column (Pharmacia Fine Chemicals, Piscataway, New Jersey; 2.5 x 40 cm) that was then eluted with saline (flow rate, 60 ml/hr; fraction size, 1.2 ml).
  • the void volume was determined by blue dextran, and 100 ul
  • An intravenous challenge dose of cell wall is administered to the rabbits and after 4 hours the rabbits are intravenously injected with one of the following tracers: 125I albumin (o.lmC ⁇ /kg), 3H peni.ci.lli.n (1 mCi/kg) or 4kDa fluoresceinated dextran (50 mg/kg) . After 30 minutes CSF and serum are collected. The ratio of the amount of tracer in CSF to that in the serum increases if the permeability of the BBB is enhanced. These values are then compared to values obtained with control rabbits injected with saline followed by IV injection with the tracer.
  • the CSF radioactivity is quantitated in a scintillation counter and fluorescein is quantitated in a spectrofluorimeter.
  • Total CSF protein concentrations are measured by the BCA method (Pierce Chemical Co.) and are increased when serum protein leaks into the CSF.
  • B - Assay procedure for visualizing the enhanced appearance of fluoresceinated dextrans in the interstitial fluid of the brain In this procedure rabbits are not placed in a frame or anesthetized. The rabbits are injected intravenously with a challenge dose of cell wall. After 4 hours, the rabbits are injected intravenously with fluoresceinated dextran tracer. 30 min later the rabbits are euthanized, a craniotomy is performed and the brain removed and examined under ultraviolet light. Fluorescence visible to the eye on the surface of the brain indicates leakage of the tracer from the intravascular space to the intersitital fluid of the brain. The animal model was adapted from Mayhan and Heistad, supra.
  • This example illustrates the increase in serum protein in the CSF resulting from enhanced permeability of the BBB in accordance with the present invention.
  • IV saline negative control
  • IC walls positive control
  • IV cell walls 3.3
  • This example demonstrates the enhanced appearance of 3H peni.ci.lli.n i.n CSF by the method of this invention.
  • This therapeutic agent is known to have poor permeability across the BBB.
  • Ten rabbits were injected intravenously with purified cell wall (Preparation A) and 6 rabbits received saline solution intravenously. After 4 hours, all the rabbits were injected intravenously with 3H peni.ci.lli.n (1 Ci/kg) . The results are shown in Table 2.
  • CSF by at least 2 fold in a dose-dependent manner.
  • concentration of penicillin in CSF in wall- treated rabbits (1/3 of serum level) correlates closely with the amount of penicillin which appears in the CSF of human patients with infla med meninges; the value for the saline control (5% of serum level) also correlates with values in humans, with intact, non-permeable BBB.
  • This example illustrates the increased amount of fluoresceinated dextran in CSF by the method of this invention.
  • Ten rabbits were injected intravenously with 0.5 mg/kg of purified cell wall (Preparation A) and 6 other rabbits received saline solution intravenously. After 4 hours, all animals were injected intravenously with one size of fluoresceinated dextran (4, 40 or 70 kDa) .
  • the amount of the fluorescein tracer in the CSF was quantified by spectrofluorimetry. The results are shown in Table 3.
  • Table 3 indicates that small sized molecules (4 kDa) and mid-sized molecules (40 kDa) cross the BBB after IV injection of cell walls, but do not cross after IV injection of saline.
  • larger molecular size (70 kDa) tracers do not seem to penetrate the BBB with the given dose of IV cell wall injection indicating that the degree of permeability may be a function of the size of the tracer. This may reflect any of a number of factors and is not a limitation of this invention since radiolabeled albumin (approximately 70K) crosses the BBB after cell wall administration (Example 1) .
  • the detection limits of the two assays differs.
  • a larger dosage of cell wall may be necessary to induce sufficient permeability of the
  • This example illustrates the increased amount of fluoresceinated dextran in interstitial fluid (IF) of the brain by this invention.
  • Negative control Four rabbits were injected intravenously with saline. After 4 hours, two rabbits received IV injection of 4kDa fluoresceinated dextran (50 mg/kg) while the other two were injected with 70kDa of this tracer. Thirty minutes later, ultraviolet inspection of the brain by the assay procedure hereinbefore described did not show the presence of these tracers in the IF. This indicates that BBB was not permeabilized.
  • This example illustrates the dose response capabilities of the pneumococcal cell wall.
  • Cell wall of the bacterium Streptococcus pneumoniae was purified as in Preparation A, supra.
  • the purified cell wall was injected intravenously to rabbits at doses of 0.5, 0.3, 0.05 and 0.005 mg/kg body weight (2 rabbits each dose) . It was found that the IV injection of this purified cell wall induced fluorescence of the IF (4 kDa marker) at the three higher dosages, but not at 0.005 mg/kg.
  • This example illustrates the timing of opening of the BBB after intravenous injection of the cell wall.
  • This example illustrates the activity of CWF structural variants in opening the BBB.
  • This example illustrates that the permeability of the BB induced by CWF is evident on histologic examination of brain tissue.
  • the CWF may be used in the form of a solution thereof in a pharmaceutically acceptable carrier such as isotonic aqueous saline or dextrose.
  • a pharmaceutically acceptable carrier such as isotonic aqueous saline or dextrose.
  • the dosage level of the CWF can vary from about 0.05% mg/kg body weight to about 50 mg/kg body weight.

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  • Health & Medical Sciences (AREA)
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Abstract

L'invention concerne un procédé permettant d'augmenter la perméabilité de la barrière hémato-encéphalique afin de pouvoir introduire des agents thérapeutiques utiles au niveau clinique, de manière qu'ils passent du flux sanguin jusque dans le cerveau et dans le liquide cérébro-spinal. Le procédé comprend l'injection parentérale de la membrane d'une cellule purifiée d'eubactérie ou de fragments de celle-ci, telle que le Streptococcus pneumoniae, cela permettant de stimuler la montée des substances thérapeutiques dans le cerveau ou dans le liquide cérébro-spinal.
PCT/US1991/002533 1990-04-13 1991-04-12 Procedes et compositions permettant de rendre permeable, de maniere reversible, la barriere hemato-encephalique WO1991016064A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP91508041A JPH05506660A (ja) 1990-04-13 1991-04-12 血液脳関門を可逆的に透過させるための方法および組成物

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
US50931990A 1990-04-13 1990-04-13
US509,319 1990-04-13
US51555290A 1990-04-27 1990-04-27
US515,552 1990-04-27
CA002081838A CA2081838A1 (fr) 1990-04-13 1992-10-30 Methodes et compositions servant a la permeabilisation reversible de la barriere hemato-encephalique

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WO1991016064A1 true WO1991016064A1 (fr) 1991-10-31

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EP (1) EP0527810A4 (fr)
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6117454A (en) * 1994-02-28 2000-09-12 Medinova Medical Consulting Gmbh Drug targeting to the nervous system by nanoparticles

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4866042A (en) * 1987-11-18 1989-09-12 Neuwelt Edward A Method for the delivery of genetic material across the blood brain barrier

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4866042A (en) * 1987-11-18 1989-09-12 Neuwelt Edward A Method for the delivery of genetic material across the blood brain barrier

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP0527810A4 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6117454A (en) * 1994-02-28 2000-09-12 Medinova Medical Consulting Gmbh Drug targeting to the nervous system by nanoparticles

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EP0527810A1 (fr) 1993-02-24
EP0527810A4 (en) 1993-06-16
CA2080376A1 (fr) 1991-10-14
CA2081838A1 (fr) 1994-05-01

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