WO1991016064A1

WO1991016064A1 - Methods and compositions for reversibly permeabilizing the blood brain barrier

Info

Publication number: WO1991016064A1
Application number: PCT/US1991/002533
Authority: WO
Inventors: Alexander Tomasz; Elaine Tuomanen
Original assignee: The Rockefeller University
Priority date: 1990-04-13
Filing date: 1991-04-12
Publication date: 1991-10-31
Also published as: EP0527810A1; EP0527810A4; CA2080376A1; CA2081838A1

Abstract

A method is provided for enhancing the permeability of the BBB in order to permit the introduction of clinically useful therapeutic agents from the blood stream into the brain and the CSF. The method comprises parenteral injection of purified cell wall or fragments thereof of eubacteria such as Streptococcus pneumoniae, thereby to enhance the uptake of the therapeutic substances into the brain and CSF.

Description

METHODS AND COMPOSITIONS FOR REVERSIBLY PERMEABILIZING THE BLOOD BRAIN BARRIER

RELATED APPLICATION

_*

This application is a continuation in part application of copending application serial number 5 (Attorney's Docket 18258) filed April 13, 1990.

FIELD OF THE INVENTION

This invention relates to permeabilization of the blood brain barrier (BBB) . It is particularly related to a method of enhancing the permeability of

10 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

15 of the brain and the cerebrospinal fluid (CSF).

More specifically, 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.

BACKGROUND OF THE INVENTION

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. While in other organs the cells of the endothelial layer have gaps and channels between them that run all the way through the layer, such channels are lacking in the cerebral capillary endothelium which is unique both in terms of the anatomically tight junctions between its cells and in terms of the rarity of pinocytic vesicles that can be frequently seen in other endothelia ("The Blood-Brain Barrier", by Gary W. Goldstein and Lorris Betz, Scientific American [1986], pages 74-83, the description of which is fully incorporated herein by reference) .

In the pathological state of bacterial meningitis 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.

In a normal (healthy) state, only substances capable of traversing the BBB can enter the brain and such substances tend to be relatively hydrophobic (lipid-like) . Substances which are hydrophylic (water-soluble) penetrate the BBB much less effectively or not at all. Such water-soluble and poorly penetrating substances encompass a whole range of molecules extending from molecules as large as albumin to ions as small as sodium, as well as chemotherapeutic agents, drugs, diagnostic imaging compounds and proteins of potential therapeutic use. While some therapeutic agents have sufficient degrees of lipid-solubility to penetrate the BBB, the great majority of drugs (e.g., penicillin) and other therapeutically useful substances have limited lipid-solubility, hence cannot penetrate the BBB well. This poor permeability of BBB by many potentially useful drugs poses a severe limitation on the treatment of diseases of the brain tissue and CSF. It is therefore of paramount clinical significance to develop products and methods which would "open" the BBB and allow access to the brain tissues and CSF by agents which are known to be effective in treating or diagnosing brain disorders but which, on their own, would not be able to traverse the BBB.

Goldstein and Betz in their aforementioned article briefly mention two modes of administering medicines to the brain. According to one mode, 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. However, this latter method can easily lead to serious mechanical injury during introduction of the needle.

United States Patent No. 4,866,042 issued September 12, 1989 to Edward A. Neuwelt describes a method for treating genetic and neurodegenerative diseases by the introduction of a corrective genetic material into the brain. The method described comprises inserting a genetic material into a virus, chemically disrupting the BBB so as to increase its permeability, administering the genetic material- containing virus into the bloodstream for incorporation into the cellular tissues of the brain and allowing the virus to deliver the genetic material into the cellular tissues of the brain. As stated in said patent, the BBB is disrupted by altering the inter-endothelial layer comprising the BBB, preferably by chemical agents. The patent of Neuwelt does not deal with the issue of BBB permeability. It is designed to bypass problems which are associated with the transplantation and enzyme replacement techniques. Thus, the Neuwelt patent does not spell out any novel methodology as to the method of disruption of BBB, which is the object of this invention.

In an article entitle "Permeability of Blood-

Brain Barrier to Various Sized Molecules" by William G. Mayhan and Donald M. Heistand, Am. J. Physiol. 248 (1985), pages 712-718, the authors report that acute hypertension produces transient disruption of the BBB.

Notwithstanding the efforts of prior workers in this field, there is not, so far as is known, a clinically safe, effective and reversible method of enhancing the permeability of the BBB to permit the passage of useful and corrective therapeutic agents into the brain in order to treat brain disorders. There is, therefore, a dire need for a therapeutic procedure for reversibly opening and closing the BBB so that intravenously administered therapeutic agents can cross the BBB.

Tomasz, Tuomanen et al. have conducted extensive studies of the mechanism of meningitic disease caused by the bacterium Streptococcus pneumoniae and have shown that the purified cell wall of that microorganism, and several subcomponents of the cell wall as well as the so- called Forssman antigen (a pneumococcal surface polymer related in chemical structure to a wall component) were all capable of causing a breakdown in the BBB when introduced into the CSF space in an experimental model of disease. Tomasz, Toumanen et al. obtained similar findings using cell walls prepared from a variety of other bacteria as well. All of these substances, when introduced intracisternally into the CSF space will permit the passage of cellular and serum components of the blood through the blood brain barrier and induce other signs and symptoms of meningitis associated with the living organism, including fever, opisthotonus, seizures, paralysis and death. See, for example, the following publications, all of which are incorporated herein by reference:

1. Quigliarello et al_^. , J. Clin. Invest., vol. 77 (1986), pages 1084-1095.

2. Tomasz, Review of Infectious Diseases, vol. 3, no. 2, March-April 1981, pages 190-211. 3. Garcia-Bustos et al. , J. Biol. Chemistry, vol. 262 (1987), pages 15400-15405. 4. Tuomanen et aJL. , J. Infect. Dis., vol. 151 (1985), pages 859-868. 5. Tuomanen, Proceedings of the 18th Workshop Conference, Hoechst, Schloss Ringberg, October 1987, pages 79-90.

6. Tuomanen et al, J. Infect. Dis. vol. 151 (1985), pages 535-540.

It is an object of this invention to provide a method for permeabilizing the BBB.

It is a further object of this invention to provide a method for enhancing the permeability of the BBB in order to permit the passage of therapeutic agents, corrective drugs, diagnostic agents, imaging agents and other useful compounds and products including, for example, transformed viruses functioning as vectors carrying DNA for correcting genetic brain abnormalities (hereinafter, for convenience, collectively referred to as therapeutic agents) into the brain and the CSF.

It is yet another object of this invention to provide a method for enhancing permeability of the BBB and morphologically similar body barriers which is reversible and non-injurious.

It is also an object of this invention to provide for the administration of cell wall fragments (CWF) to humans or other mammals afflicted with brain disease or disorder to permeabilize the BBB so as to permit the introduction of corrective therapeutic agents into the brain and the CSF for the treatment of tumors, infections, degenerative diseases, autoimmune diseases, multiple sclerosis and other diseases. The foregoing and other objects and features of the present invention will be more fully appreciated from the ensuing description.

SUMMARY OF THE INVENTION

In accordance with this invention, 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".

For convenience, the invention will be described principally with reference to 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.

In some aspects of this invention it appears that 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.

DESCRIPTION OF THE DRAWING

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.

DETAILED DESCRIPTION OF THE INVENTION

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. In the invention to be described here, 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.

As discussed in references 2 and 3, 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. As stated above and as known to those skilled in the art, 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. As used in this description and claims, 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.

To assist in the description of this invention, the preparation of purified pneumococcal cell wall and some of its fragments is described below. The preparations, namely Preparation A and Preparation F, are described in detail in reference 4.

Preparation "A" - purified cell wall with choline-containinq teichoic acids: Streptococcus pneumoniae R₆ (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,

Minnesota) and shaken at maximum amplitude at 4°C for 3 hours. An airspace equal to the total volume of the suspension was kept in the cuvettes to allow efficient disruption. The glass beads were then 10 separated out by sedimentation, the suspension was centrifuged at 3,000 g for 3 minutes (Sorvall RC2B; Sorvall, Newton, Conn.) to remove unbroken cells, and the suspension was centrifuged at 10,000 g for 30 minutes to sediment out the cell wall material. 15 Inspection by phase-contrast microscopy revealed only amorphous debris in such preparation. This crude cell wall material was extracted with 2% sodium docecyl sulfate (SDS) at 90-100°C for 30 minutes. The detergent was removed with six cycles 20 of washings by centrifugation in distilled water.

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 CaCl₂ 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 detergent was removed by 8 to 10 cycles of washing, first in 1M NaCl solution and then in distilled water, and the purified cell wall was lyophilized. Preparation "F" - cell wall de ration products. Amidase product: peptide-poor glycan-teichoic acid complex: Two batches of pneumococcal cell wall (80 mg. of preparation A) were each resuspended in 3.5 ml of saline containing lOmM of K_HP0 (pH 7.4; SP) . One batch received 100 ul [ 3H] methyl cholme-

5 labelled cell wall (about 10 cpm total) and the other, 100 ul [ 3H] lysine-labelled cell wall as tracers. Each suspension received 0.3 g of pneumococcal autolysin (400 ul containing approximately four units of enzyme activity) prepared as in the method described by A. Tomasz et al, "The Physiological Functions of Teichoic acids, J. Supramol Stud. (1975), page 3, the disclosure of which is fully incorporated herein by reference.

The suspensions were incubated at 30°C for 48 hours during which time enzymatic solubilizations was monitored as described by J.V. Holtje et al. Purification of the pneumococcal N-acetylmuramyl-L saline Amidase to Biochemical Homogeneity, J. Biol. Chem., Vol 251 (1976), pages 4199-4207, the disclosure of which is fully incorporated herein by reference. Pneumococcal autolysin is N- acetylmuramic acid-L-alumni amidase capable of degrading the pneumococcal cell wall to two types of components separable on the basis of their different molecular sizes. 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, and 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

^* 5 portions of the fractions were assayed for radioactivity in 4 ml of Ultrafleur by using a

* scintillation spectrometer (Nuclear Chicago, Hartsdale, New York) . The fractions representing the high molecular weight material (glycan-teichoic

10 acid complex) were pooled, lyophilized, dissolved in distilled water, desalted by passing through a column of Sephadex G-10 (2.5 x 30cm) and lyophilized. The yield was 11.91 g. This material was found to be a powerful inducer of inflammation

15 when introduced intrathecally in the rabbit model of experimental meningitis.

As will hereinafter be described, purified cell wall at doses of 0.5, 0.3 and 0.05 mg/kg isolated as in Preparation "A" above induced fluorescence of the

20 brain of rabbits after IV administration of a fluorescent tagged-4-kDa tracer. Fluorescence was lost at 0.005 mg/kg body weight. The same preparation also enhanced the influx of penicillin, radioactively-labeled albumin and fluorescent tracer

25 substances into the CSF of rabbits.

The following examples illustrate in more detail that the intravenous injection of purified pneumococcal cell wall enhances permeability of the BBB and permits the passage of substances which 30 normally cannot penetrate or permeate only poorly through the BBB. Two types of assays were used to establish disruption of the BBB by the IV administration of the pneumococcal cell wall fragment. Assay procedures A and B are described below.

A. Assay procedure for measuring enhanced appearance of 125 I albumi.n, 3H peni.ci.lli.n and 4kDa fluoresceinated dextran in CSF: (The assay procedure described herein is a variant of the procedure described by E. Tuomanen et al. in reference 6.) Rabbits are anesthetized and placed in a stereotaxic frame. A spinal needle is introduced into their cisterna magna for sequential sampling of CSF. 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.lmCι/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.

Example 1

This example illustrates the increase in serum protein in the CSF resulting from enhanced permeability of the BBB in accordance with the present invention.

Two rabbits were injected intravenously with 0.5 mg/kg body weight of purified cell wall (Preparation A) . Another two rabbits were injected intravenously with saline solution (negative control) and the last two rabbits were injected intracisternally with the same cell wall preparation

(positive control) . After 4 hours all six rabbits were i .nj.ected i.ntravenously wi.th 125I albumin (0.1 mCi/kg) . The results are shown in Table I below. Table 1

Amount of 125I albumin

Substance injected in CSF as % serum level

IV saline (negative control) 1.8 IC walls (positive control) 3.4 IV cell walls 3.3

The results in Table 1 indicate that the administration of saline had no effect on permeabilizing the BBB but the IV cell walls opened the BBB to the same magnitude as observed during the intense inflammatory response observed following intracisternal inoculation of cell walls (the latter mimics meningitis) . This influx of protein into the CSF, associated with IV inoculation of cell walls, was not accompanied by the appearance of leukocytes in the CSF. This is thought to be an advantage since leukocytes contribute to BBB injury which leads to permanent deficits in neuronal function.

Example 2

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.

Table 2

Challenge Dose Amount of H penicillin Material in CSF, as % serum level

saline 1 ml/kg 9.6 + 1.3 cell wall 0.1 mg/kg 17.2 + 0.3 cell wall 0.01 mg/kg 20.0 + 3.8

As shown in Table 2, the administration of cell wall increases the 3H penicillin penetration into

CSF by at least 2 fold in a dose-dependent manner. The 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.

Example 3

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

Challenge Fluorescein Amount of fluorescence Material Tracer in CSF, as % serum level

All cell wall treated animals showed increased total protein in CSF (mean 1.8 + 0.4 mg/ml) as compared to controls (mean 0.7 + 0.01 mg/ml) indicating permeabilized BBB.

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. Under the conditions of this experiment, 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

BBB to show a positive result for large molecules in the assay as described in this examples.

Example 4

This example illustrates the increased amount of fluoresceinated dextran in interstitial fluid (IF) of the brain by this invention.

A. Control experiments:

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.

Positive control. In a companion series of experiments, four rabbits with established pneumococcal meningitis each received either 4, 20, 40 or 70 kDa fluoresceinated dextran (50 mg/kg) . After 30 minutes, the brain of each rabbit was examined visually by ultraviolet light. All showed the presence of fluoresceinated dextran indicating that all sized tracers had passed into the IF across the BBB which was permeabilized by meningeal infection.

B. Effect of IV injection of purified cell wall (Preparation A) : Sixteen rabbits were each injected intravenously with 0.5 mg/kg of pneumococcal cell wall. After 4 hours, 4 rabbits each were injected intravenously with fluoresceinated dextran of 4, 20, 40 or 70 kDa (50 mg/kg) . visual inspection of the brain under ultraviolet light showed bright fluorescence of the brains after 4 or 20 kDa tracer, small but detectable fluorescence after 40 kDa tracer, and no fluorescence after 70 kDa tracer. This indicates the 4, 20, and, to a lesser extent, 40 kDa tracers cross the BBB whereas the 70 kDa tracer could not penetrate the BBB even after cell wall administration. These results are consistent with the results obtained in the control experiments of Section A above and indicate that the permeability of the BBB to large molecules occurs to a greater extent in meningitis than following cell walls IV. Permeability changes to smaller molecules (<40 kDa) occurs in both cases.

Example 5

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.

Example 6

This example illustrates the timing of opening of the BBB after intravenous injection of the cell wall.

Eighteen rabbits were injected intravenously each with 0.5 mg/kg body weight of Preparation A. At 1, 2, 3, 4, 6, 8, 20, 24 and 30 hours thereafter, 4 kDa fluorescein-labeled tracer was injected intravenously into pairs of rabbits and after 30 minutes the brains of the animals were examined under ultraviolet light.

In 6 rabbits, one each at time points 1 to 8 hours, CSF was obtained for protein determination and fluorescence detections. The results are shown in Table IV.

TABLE IV

Time post CWF injection Pre 1 2 3 4 hours

fluorescence brain IF — — — — + fluorescence CSF — — — — + CSF protein concentration

(mg/ml) 0.7 0.7 0.7 0.9 1.6

Time post CWF injection 6 8 20 24 30 hours

fluorescence brain IF + + + fluorescence CSF + + CSF protein concentration

(mg/ml) 2.2 1.9

This example indicates that the BBB becomes permeabilized at about 4 hours post-infusion of the cell wall and the barrier remains open for about 20 hours. The opening, therefore, is reversible.

Example 7

This example illustrates the activity of CWF structural variants in opening the BBB.

Two control rabbits received saline IV and 6 rabbits were challenged with 0.05 mg/kg IV of CWF containing ethanolamine (ECWF) in place of choline in the teichoic acid (Preparation B of ref. 4) , and 5 rabbits received hydrolyzed cell wall (Preparation

F, supra). At 4, 8 or 24 hours later, 4 kDa fluorescein marker and (1 mCi/kg) 3H-peni.ci.llm. were injected IV to pairs of animals. CSF and serum samples were taken 30 min later, the animals were then euthanized and the brain and CSF examined under ultraviolet light. The results are shown in Table

V.

TABLE V

This example indicates that 2 chemical variants of CWF also induce BBB permeability as shown by the appearance of fluorescent marker in brain IF and CSF and enhanced permeability of 3H penicillin from serum into CSF. Permeability may be reversed sooner after treatment with CWF variants than with CWF.

Example 8

This example illustrates that the permeability of the BB induced by CWF is evident on histologic examination of brain tissue.

Two rabbits were examined by this technique - one received saline IV (control) and the other 1 mg of cell wall Preparation A supra. Four hours later the animals received an IV infusion of horseradish peroxidase. Ten minutes later both rabbits were euthanized, the brains removed and cerebral capillaries were isolated and fixed in glutaraldehyde (ref. 1) . The fixed tissue was examined by electron microscopy and the distribution of the horseradish peroxidase stain determined. In the control rabbit, the stain remained within the capillary lumen. In contrast, in the CWF-treated animal, the stain leaked out of the capillary into the brain IF indicating a permeabilized BBB.

It can be appreciated from the foregoing detailed description that some variations may be made in the method described herein which nevertheless are contemplated by the present invention. For example, the CWF may be used in the form of a solution thereof in a pharmaceutically acceptable carrier such as isotonic aqueous saline or dextrose. Also, the dosage level of the CWF can vary from about 0.05% mg/kg body weight to about 50 mg/kg body weight.

Claims

WHAT IS CLAIMED IS:

1. A method for permeabilizing the blood brain barrier of mammals in need of such permeabilization to permit passage of therapeutic agents into the brain and the cerebrospinal fluid which method comprises parenteral administration of an amount which is effective to cause such permeabilization of a cell wall component selected from the group consisting of purified cell wall or cell wall fragments of eubacteria having the ability when injected intravenously to permit the passage of components of the blood through the blood brain barrier.

2. A method for the administration of a corrective therapeutic agent into the brain and the cerebrospinal fluid of mammals in need of such agent, which comprises parenteral injection in an amount which is effective to permeabilize the blood brain barrier of a cell wall component selected from the group consisting of purified cell wall or cell wall fragments of eubacteria, in an amount which is effective to permeabilize the blood brain barrier having the ability when injected intravenously to permit the passage of components of the blood through the blood brain barrier, together with an amount of the therapeutic agent which is sufficient to cause such correction.

3. A method as in claim 1 wherein said mammal is a human.

4. A method as in claim 2 wherein said mammal is a human.

5. A method as in claim 1 wherein said cell wall component is administered at a dosage level of from about 0.05 mg/kg body weight to about 50 mg/kg body.

6. A method as in claim 2 wherein said cell wall component is administered at a dosage level of from about 0.05 mg/kg body weight to about 50 mg/kg body weight.

7. A method as in claim 2 wherein said therapeutic agent is administered from about 4 to about 20 hours after injecting said cell wall fragment.

8. A method as in claim 1 or 2 wherein said parenteral injection is intravenous.

9. A method as in claim 2 wherein the corrective therapeutic agent is a diagnostic agent.

10. A method as in claim 2 wherein the corrective therapeutic agent is an imaging agent.

11. A method as in claim 3 wherein said therapeutic agent is administered from about 4 to about 20 hours after injecting said cell wall fragment.

12. A method as in claim 4 wherein said therapeutic agent is administered from about 2 to about 20 hours after injecting said cell wall fragment.

13. A method as in claim 1, 2, 3, 4, 5, 6, 7,

10, 11 and 12 wherein the eubacteria is Streptococcus pneumonia.

14. A method as in claim 1, 2, 3, 4, 5, 6, 7, 10, 11 and 12 wherein the eubacteria is Micrococcus lysodekticus.

15. A method as in claim 1, 2, 3, 4, 5, 6, 7, 10, 11 and 12 wherein the eubacteria is Bacillus subtilis.

16. A method as in claim 1, 2, 3, 4, 5, 6, 7, 10, 11 and 12 wherein the eubacteria is Escherichia coli.