METHOD FOR INCREASING BLOOD-BRAIN BARRIER PERMEABILITY

Abstract: The present invention pertains to a method for increasing the permeability of the blood-brain barrier of a host to a molecule present in the host's bloodstream. The method comprises intravenous co-administration to a host of an effective amount of a bradykinin agonist of blood-brain barrier permeability.

Description: METH0D_F0R_I CREA 1NG_BL00D_ BRA1N BARRIER PERMEABILITY As our understanding of the nervous system and its related disorders increases, a wider range of therapeutic and diagnostic agents will become available. Once these agents have been identified, it will be necessary to deliver them to sites of diseased tissue in the central nervous system. Unfortunately, the existence of the blood-brain barrier limits the free passage of many types of molecules from the blood to cells of the central nervous system. The physiological basis for the blood-brain barrier is the brain capillaries, which are comprised of endo- thelial cells (Goldstein, «ϊ.t_a 1__ , S_c ± eτι ±fi.£_Ameri.£an , 255: 74-83 (1986); Pardridge, W.M. , Endocrin J __Rev_, 7: 314-330 (1986)). These endothelial cells are different from those found in other tissues of the body. In particular, they form complex tight junctions between themselves. The actual blood-brain barrier is formed by these high-resistance tight intercellular junctions which form a continuous wall against the passive movement of many molecules from the blood to the brain. These cells are also different in that they have few pinocytotic vesicles, which in other tissues allow somewhat un- selective transport across the capillary wall. In addition, continuous gaps or channels running through the cells, which woulc allow unrestrained passage, are alis p nt One function of the blood-brain barrier is to protect the brain from fluctuations in blood chemistry. However, this isolation of the brain from the bloodstream is not complete. There does exist an exchange of nutrients and waste products. The presence of specific transport systems within the capillary endothelial cells assures that the brain receives, in a controlled manner, all of the compounds required for normal growth and function. The obstacle presented by the blood-brain barrier is that, in the process of protecting • the brain, it excludes many potentially useful therapeutic and diagnostic agents. There are several techniques that either physically break through the blood-brain barrier or circumvent it to deliver therapeutic or diagnostic agents. Among these are intrathecal injections, surgical implants, and osmotic techniques. Intrathecal injection allows administration of agents directly into the brain ventricles and spinal fluid by puncturing the membranes surrounding the brain. Sustained delivery of agents directly into the spinal fluid can be attained by the use of infusion pumps that are implanted surgically. These spinal fluid delivery techniques are used to treat brain cancers, infections, inflammation and pain. However, they often do not result in deep penetration of substances into the brain. Clinicians prefer to avoid intrathecal injections because they frequently are ineffective and can be dangerous. Substances injected intrathecally are dis¬ tributed unevenly, slowly and incompletely in the brain. Since the volume of the spinal fluid is small, increases in intracerebral pressure can occur with repeated in¬ jections. Furthermore, improper needle or catheter placement can result in seizure, bleeding, encephalitis and a variety of other severe side effects. An osmotic approach has been used by Dr. Edward Neuwelt at the University of Oregon to deliver chemo- therapeutics and imaging antibodies to tumors in the brain. (Neuwelt, E.A. , Implication of the Blood-Brain Barrier and its Manipulation, Vols 1 & 2, Plenum Press, N.Y. (1989)) This technique involves an arterial injection of a bolus of a hypertonic mannitol solution. The osmotic differential exerted by the mannitol causes the endothelial cells forming the barrier to shrink, opening gaps between them for a brief period. During this period, the drug is administered into the arterial system and is carried directly into the brain. The osmotic approach demonstrates that once past the barrier, therapeutic agents can be effectively distributed throughout the brain. Because of the many risks involved, a 24- to 48- hour period in an intensive care unit is necessary following osmotic treatment. Mannitol can cause perma¬ nent damage (including blindness) to the eye. If the barrier is permeable for too long, brain edema results. Cells of the brain also can be damaged when neurotoxic substances in the blood, not generally accessible to the brain, are able to cross the barrier. Finally, there is a significant incidence of seizures in patients during and after the procedure. ^ummarγ_ f_the_Invention The present invention pertains to a method of increasing the permeability of the blood-brain barrier of a host to a molecule contained in the host's bloodstream. This method comprises intravenous co-administration to the host of an effective amount of a bradykinin agonist of blood-brain barrier permeability. The molecule to be delivered to the brain can be either an endogenous molecule or an exogenous molecule that is co-administered sequentially or simultaneously with the bradykinin agonist of blood-brain barrier permeability. An advantage of the present invention is that it provides a practical means of increasing the permeability of the blood-brain barrier by the intravenous adminis¬ tration of a bradykinin agonist of blood-brain barrier permeability while co-administering a molecule of thera¬ peutic, prophylactic cr diagnostic value. In contrast to osmotic treatment or intrathecal delivery, intravenous injection of a bradykinin agonist of blood-brain barrier permeability is significantly less traumatic, does not require surgery and is unlikely to necessitate anesthesia. Furthermore, in contrast to the osmotic techniques which allow entry into the brain of a large variety of molecules present in the blood, including protein, the bradykinin agonist of blood-brain barrier permeability preferentially induces passage through the blood-brain barrier of small molecules. The intravenous route of administration of a bradykinin agonist of blood-brain barrier permeability offers a number of significant advantages over other routes of administration (subcutaneous or intramuscular injection, as well as the more drastic measures of intrathecal or carotid artery injection) . For example, the intravenous route of administration affords a more convenient method for providing rapid drug action. The intravenous route also offers better control over the rate of administration of drugs; prolonged action can be provided by administering an infusion either inter¬ mittently or over a prolonged period of time. Also, slow intravenous administration of a drug permits termination of the infusion if sensitivity occurs. In addition, certain drugs, because of their irritating properties, cause pain and trauma when given by the intramuscular or subcu aneous route and must be given intravenously. Additionally, some drugs cannot be absorbed by any other route or are unstable in the presence of gastric juices. The intravenous route also affords a means of adminis¬ tration for the patient who cannot tolerate fluids and drugs by the gastrointestinal route. Delivering thera¬ peutic and diagnostic agents into the brain by an intra¬ venous technique is unique and will improve dramatically and immediately the clinician's ability to understand, diagnose and treat diseases of the central nervous system . 6- -----li£-_-^1£Ξi£-£i£-S_ -t—-------------∑---H-Ξ-----i Figure 1 is a graphic representation of brain uptake (μl/g) of sucrose and bovine serum albumin (BSA) co- administered with bradykinin at several doses of bradykinin (μg/mouse) . Figure 2 is a graphic representation of the time course of brain uptake of sucrose after administration of a specific amount (10 μg ) of a bradykinin analogue into different animals. Figure 3 is a graphic representation of the time course of brain uptake of sucrose after the adminis¬ tration of a specific amount (100 μg ) of a bradykinin analogue . Figure 4a is a graphic representation of brain uptake of sucrose, displayed as percent of injected dose, following the administration of bradykinin and captopril or bradykinin analogues . Figure 4b is a graphic representation of brain uptake of sucrose, displayed as percent of maximum uptake, following the administration of bradykinin and captopril or bradykinin analogues. Figure 5 is a histogram which illustrates the effect of co-administered bradykinin on the antinociceptive activity of loperamide in the mouse tail flick assay. Figure 6 is a histogram which illustrates the effect of a co-administered bradykinin analogue on the anti¬ nociceptive activity of loperamide in the mouse tail flick assay. Figure 7 is a graphic representation of the effects of a dopamine analogue, diacetyldopamine (AcDA) , on the motor activity of rats when administered alone, with bradykinin or with bradykinin and haloperidol (Haldol) . Figure 8 is a graphic representation of the effects of no treatment (control), treatment with cisplatin, treatment with captopril and co-administered bradykinin, and treatment with cisplatin, captopril and co- administered bradykinin in the survival time (days) of rats implanted with a brain tumor, with treatments occuring at days 4 and 6 after tumor implantation. Figure 9 is a graphic representation of the effects of no treatment, treatment with cisplatin and treatment with cisplatin, captopril and co-administered bradykinin on survival time (days) of rats implanted with a brain tumor, with treatment period lasting from day 4 to day 14. Figure 10 is a graphic representation of the effects of no treatment; treatment with cisplatin; treatment with bradykinin, captopril and cisplatin; and treatment with a bradykinin analogue and cisplatin on survival time (days) of rats implanted with a brain tumor. Figure 11 is a graphic representation of the brain uptake of an imaging agent intravenously injected 3 minutes after either saline or one of three different amounts of bradykinin analogue was intravenously injected into rats . Figure 12 is a histogram which illustrates the effect of a bradykinin analogue co-administered with an imaging agent on the uptake of the imaging agent into the brain at various times after injection. Figure 13 is a graphic representation of the effects of a dopamine receptor antagonist (domperidone) co- administered with a bradykinin analogue on apomorphine- induced motor activity of rats. Figure 14 is a graphic representation of the effects of an angiotensin II analogue co-administered with a bradykinin analogue on drinking behavior in rats. Figure 15 is a histogram which illustrates the effects of an angiotensin II analogue and inhibitor co-administered with a bradykinin analogue on drinking behavior in rats. D ta^]Le_d_De cri.£ti n_of_ he_Jnvent^£n This invention relates to a method for increasing the permeability of the blood-brain barrier of a host to a molecule present in the host's bloodstream. The increase in permeability of the blood-brain barrier is accomplished by intravenously co-administering a bradykinin agonist of blood-brain barrier permeability to the host. By the phrase "bradykinin agonist of blood- brain barrier permeability" is meant a compound that elicits an increase in blood-brain barrier permeability in the manner of bradykinin. The host can be any animal which possesses a central nervous system (i.e. , a brain). Examples of hosts include mammals, such as humans, domestic animals (e.g, dog, cat, cow or horse) and animals intended for experi¬ mental purposes (e.g. , mice, rats, rabbits). The molecule in the host's bloodstream can be exogenous to the host. For example, it can be a neuro- pharmaceutical agent which has a therapeutic or pro¬ phylactic effect on a neurological disorder. Examples of neurological disorders include cancer (e.g. , brain tumors), Autoimmune Deficiency Syndrome (AIDS), epilepsy, Parkinson's disease, multiple sclerosis, neurodegener- ative disease, trauma, depression, Alzheimer's disease, migraine, pain, or a seizure disorder. Classes of neuropharmaceutical agents which can be used in this invention include antimicrobial agents, antiparasitic agents, agents that act on the autonomic nervous system including adrenergic agents and catecholaminergic agents, anticonvulsants , nucleotide analogues, antineoplastic agents, anti-trauma agents, excitatory amino acids and inhibitors and other classes of agents used to treat or prevent a neurological disorder. Examples of antibiotics include amphotericin B, gentamicin sulfate, pyrimethamine and penicillin. An example of an adrenergic agent (including blockers) is atenolol. Examples of catecholaminergic agents include dopamine, diacetyldopamine and domperidone. Examples of antineoplastic agents include adriamycin, methotrexate , cyclophosphamide, etoposide, carboplatin and cisplatin. An example of an anticonvulsant which can be used is valproate. Examples of anti-trauma agents which can be used include calpain inhibitors, channel blockers, glutamate chelators and oxygen radical scavengers. Nucleotide analogues which can be used include azido thymidine (AZT) , dideoxy Inosine (ddl) and dideoxy Cytidine (ddC). The molecules in the host's bloodstream can also be diagnostic imaging or contrast agents. Examples of diagnostic agents include substances that are labelled with radioactivity, such as 99m-Tc glucoheptonate or substances used in Magnetic Resonance Imaging (MRI) procedures, such as Gadolinium-doped chelation agents (e.g. , Gd-DTPA) . The route of administration of exogenous molecules to the host's bloodstream can be parenterally by sub¬ cutaneous, intravenous or intramuscular injection or by absorption through a bodily tissue, such as the digestive tract, the respiratory system or the skin. The form in which the molecule is administered (e.g. , capsule, tablet, solution, emulsion) will depend, at least in part, on the route by which it is administered. The administration of the exogenous molecule to the host's bloodstream and the intravenous injection of bradykinin agonist of blood-brain barrier permeability can occur simultaneously or sequentially in time. For example, a therapeutic drug can be administered orally in tablet form while the intravenous administration of a bradykinin agonist of blood-brain barrier permeability is given later (e.g. 30 minutes) . This is to allow time for the drug to be absorbed in the gastrointestinal tract and taken up by the bloodstream before the agonist is given to increase the permeability of the blood-brain barrier to the drug. On the other hand, the bradykinin agonist of blood-brain barrier permeability can be administered before or at the same time as an intravenous injection of a drug. Thus, the term "co-administration" is used herein to mean that the bradykinin agonist of blood-brain barrier permeability and the exogenous molecule will be administered at times that will achieve significant concentrations in the blood producing the simultaneous effects of increasing the permeability of the blood-brain barrier and allowing the maximum passage of the exogenous molecule from the blood to the interstitial fluid of the central nervous system. In addition, the molecule to be delivered to the brain via the host's bloodstream can be endogenous to the host. That is, it can be a biological product that is naturally synthesized and produced by the host. Examples of such biological products include sugars, such as glucose, and small peptides, such as enkephalins and thyroid stimulating hormone releasing factor. Compounds are termed agonists when they increase or elicit a physiological response by interacting with structural moieties at particular anatomical regions of the body. For purposes of this invention, a compound is an agonist of blood-brain barrier permeability when it interacts with structural moieties anatomically associated with the blood-brain barrier to significantly increase the permeability of the blood-brain barrier for the molecule of interest. This effect of increased permeability of the blood-brain barrier is believed to operate through a tissue-associated receptor mediated event, probably through the B_ receptors. Other tissue- associated receptors may similarly be involved in this process to increase the permeability of the blood-brain barrier, particularly when peptides or compounds other than bradykinin or bradykinin analogues are utilized as the agonist of blood-brain barrier permeability. Examples of agonist compounds which initiate an increased permeability of the blood-brain barrier to a molecule of interest include bradykinin, bradykinin analogues, other peptides and other compounds that mimic the increase in permeability of the blood-brain barrier in the manner elicited by bradykinin. Since these substances apparently interact with tissue-associated receptors to produce an increase in the permeability of the blood- brain barrier to a molecule of interest, these substances can collectively be called "receptor mediated permea- bilizers" . Bradykinin is a naturally occurring peptide com¬ prised of nine amino acids having the following sequence: NH„ -Arginine-Proline-Proline-Glycine-Phenylalanine-Se- rine-Proline-Phenylalanine-Arginine-COOH (Lehninger, A.L. , Bi.££hemj- try_, p. 97 (1975)). A bradykinin analogue can be a structural derivative of bradykinin. Such analogues can be compounds which are derivatives of the number and/or sequence of amino acids in the bradykinin structure mentioned above which have a similar or en¬ hanced effect on permeability of the blood-brain barrier. Bradykinin analogues also include modifications to the bradykinin molecule effected by changing or chemically modifying the normal amino acids, modifying peptide bonds, adding C-terminal and/or N-terminal extensions, etc . A method for preparing bradykinin analogues is Merrifield's procedure of solid-phase peptide synthesis (Merrifield, R.B. , ÷ _Am__Chem_ L- Soc_. , 6_ : 304 (1964); Draprau, G. and Regoli, D. , £h£d _iτι_En yi£l_£ j gy . JL6J3: 263-272 (1988)). The first step in a solid-phase synthesis of bradykinin analogues is the formation of a covalent bond between the C-terminal protected amino acid of the chosen peptide sequences and the solid support or resin. The peptide chain is built up residue by residue by repetitive cycles of deprotection, during which the N-terminal Boc-protecting (N- tert-butoxycarbonyl) group is removed by trifluoroacetic acid (TFA) . This is followed by neutralization with diisopropylethylamine (DIEA) of the amino group left as a salt and coupling of the next amino acid in the sequence. The cycle is repeated until the sequence is completed. After its complete assembly, the peptide is cleaved from the resin and purified. Compounds that mimic the bradykinin-like increase in permeability of the blood-brain barrier can also be synthesized as polypeptides, purified from mixtures of naturally occurring polypeptides, be chemically modified forms of such polypeptides or be chemically synthesized structures that act as bradykinin agonists of blood-brain barrier permeability. The essential characteristic of these compounds is in their ability to elicit an increase in blood-brain barrier permeability in a manner similar to that of bradykinin. An effective amount of a bradykinin agonist of blood-brain barrier permeability is that amount which will significantly increase the blood-brain barrier permeability for the molecule of interest. In other words, it will increase the permeability of the blood- brain barrier to allow sufficient quantities of a molecule to pass from the blood to the interstitial fluid of the brain to exert a therapeutic or prophylactic effect or allow diagnostic procedures. The effective amount will be determined on an individual basis and will be based, at least in part, on consideration of the host's size, the specific disease, the severity of symptoms to be treated, the result sought and the specific bradykinin agonist of blood-brain barrier permeability used. Thus, the effective amount can be determined by one of ordinary skill in the art employing such factors and using no more than routine experi¬ mentation. The increase in permeability of the blood-brain barrier in response to a bradykinin agonist relates not only to the quantity of molecules passing from the blood to the interstitial fluid of the brain, but also, to the type of molecule to be passed from the blood to the interstitial fluid of the brain. The effect of bradykinin agonists of blood-brain barrier permeability is to preferentially increase the passage of small molecular weight substances. For example, the data listed in Example VII demonstrate that the permeability of the blood-brain barrier of mice to a 40,000 molecular weight molecule was not substantially affected while the permeability of molecules with molecular weights of 1,000 or less was statistically significantly increased. Thus, while the permeability of small molecular weight sub¬ stances is increased, the exclusion of the blood-brain barrier to substances of significantly higher molecular weight is substantially maintained. (See Example VII for further detail.) Those skilled in the art will understand that mixtures of bradykinin analogues can also be administered as agonists of blood-brain barrier permeability. For example, bradykinin plus a bradykinin analogue, such as A-7, could be administered together. In like manner, A-7 could be administered with one or more other bradykinin analogues or with bombesin or VIP or with other agonists of blood-brain barrier permeability. It will also be understood by those skilled in the art that the agonists of blood-brain barrier permeability should be non-toxic to the host. Agonists are non-toxic if a therapeutically effective amount does not result in death to the host. In a specific application of the invention, a patient afflicted with toxoplasmosis encephalitis can be treated by administering an effective amount of an antiparasitic agent, such as clindamycin or gentamycin, and co-administering an angonist of blood-brain barrier permeability. The co-administration of the agonist of blood-brain barrier permeability allows passage of the antiparasitic agent through the blood-brain barrier to treat the parasitic infection. The invention is further illustrated by the following specific examples. lHSEl£_Ii ^-f££-t_£-f_lB-£∑^Σ£- £H-£_££- Admi.n^ £r ti.£n -§-!--≤. .Z}£i£i£_£B_-§.ε i2_ϊi£-!L^ 5.£ΣHS_^i ϋSiS_XB 2• Female Balb/C mice weighing approximately 20g were employed. All solutions were prepared in sterile PBS. Intravenous injections (100 μl ) were performed in the 3 6 tail vein. H-sucrose (10 cpm) was injected as a bolus without or with bradykinin as stated. Mice were killed 10 minutes after injection. Blood was collected in heparinized tubes and a 100 μl aliquot was counted in a liquid scintillation counter after addition of 1 ml of 1% sodium dodecyl sulfate (SDS) to ensure solubilization of proteins, 300 μl 37% hydrogen peroxide as a bleaching agent, and 15 ml Aquasol-2 (Dupont) . The brain was removed, homogenized in 5 ml H_0, and 1 ml of the homo- genate was aliquoted for liquid scintillation counting (after addition of 1 ml of 1% SDS and 15 ml Aquasol-2). Relative brain uptakes were calculated as the ratios of radioactivity recovered in brain over that recovered in blood, and expressed as [radioactivity in 1 μl blood] (μl/g)/[radioactivity in lg brain] . In some experiments, 14 5 C-BSA (5 x 10 cpm) was co-injected together with 3 6 H-sucrose (10 cpm), without or with bradykinin, and the 14 3 relative brain uptakes of C-BSA and H-sucrose were calculated after double isotope scintillation counting. The results are displayed in Figure 1. As this 3 Figure shows, the relative brain uptake of H-sucrose 10 minutes after injection was significantly increased when 3 bradykinin was co-administered with H-sucrose at doses of 10, 30, 100 and 300 μg and reached a value of 33 μl/ . The threshold dose of bradykinin was 10 μg , while the two highest doses of 100 and 300 μg yielded almost identical 3 increases in relative brain uptake of H-sucrose. In contrast, bradykinin at doses of up to 100 μg had marginal effect on the relative brain uptake of C-BSA, which remained between 14 and 18 μl/g. At a bradykinin 14 dose of 300 μg , the relative brain uptake of C-BSA increased to 23 μl/g. E a.m£l _I^_ 1 _ Pharma££] 1 ££^£al_Chara£teriz ^ ati.£n_£f_th £-£i^£ _ 2_-tϊl _ϊl£H £._Bi££5-lB-E i-Q---.B Σ-Ei£.£ • The protocol for these experiments was the same as described in Example I, except as noted. Drugs were administered to the mice at the concentrations listed in Table 1. For bradykinin and bradykinin analogues, brain 3 levels of H-sucrose were determined 10 minutes after treatment. For bombesin and vasoactive intestinal 14 peptide, brain levels of C- sucrose were determined 10 minutes after treatment. The brain uptake data are expressed as mean ± s.d. and are derived from two 9 independent experiments except for the desArg - bradykinin, bombesin and vasoactive intestinal peptide results which were derived from one experiment for each peptide. Four mice were used for each different drug per experiment. The results are as follows: Table I Brain Uptake of % Dose/ sucrose Increase Treatment Mouse N ._l%_C£ntr£] 1 _ £Z £_C£ntr None (Control) 8 100 + 9 0 Bradykinin 300 μg 8 177 + 24 77 [Phe 8 Φ(CH 2 -NH)Arg 9 ]bradykinin 125 μg 8 174 + 15 74 N-acetyl[Phe 8 Φ(CH 2 -NH)Arg 9 ] bradykinin 100 μg 5 230 + 26 130 9 desArg -bradykinin 300 μg 4 90 + 15 -10 Bombesin 10 μg 4 207 ± 14 107 Vasoactive Intestinal Peptide 10 μg 4 209 ± 7 109 Bradykinin significantly increased the brain uptake 3 of H-sucrose. In addition, the bradykinin analogue and 8 9 agonist [Phe Φ(CH- -NH)Arg ]bradykinin, which is believed to be specific for B„ receptors, also increased brain uptake. This agonist differs from bradykinin in that the peptide bond between the 8th (Phe) and 9th (Arg) amino acids is replaced with the isosteric CH--NH bond in order to protect bradykinin from degradation. When this agonist is treated further with N-acetylation to give N-acetyl[Phe 8 Φ(CH_-NH)Arg ] bradykinin, the effect on brain uptake is increased to a similar extent as that obtained with bradykinin or the original analogue. This form of the analogue is also devoid of histamine- releasing activity, unlike bradykinin itself. One 9 product of bradykinin degradation is desArg -bradykinin. This degradation byproduct results from the action of kininase 1 (carboxypeptidase N) , a proteolytic enzyme, which removes the C-terminal arginine . This bradykinin analogue is believed to retain biological activity on B. receptors, but did not substantially affect the brain uptake of sucrose through the blood-brain barrier. The peptides bombesin (p Glutamate-Glutamine- Arginine-Leucine-Glycine-Asparagine-Glut mine- Tryptophan-Alanine-Valine-Glycine-Histidine-Leucine- Methionine-NH- ) and vasoactive intestinal peptide (His idine-Serine-Aspartate-Alanine-Valine- Phenylalanine-Threonine-Aspartate-Asparagine-Tyrosine- Threonine-Arginine-Leucine-Arginine-Lysine-Glutamine- Methionine-Alanine-Valine-Lysine-L sine-Tr tophan- Leucine-Asparagine-Serine-Isoleucine-Leucine- Asparagine-NH„) significantly increased the brain uptake of 14C-sucrose. This indicates that these peptides are receptor mediated permeabilizers . ?ϋ£5El-!_IIL X2iihe i. _£f_th _B££ 4_ 1 MeTy_r_ tCH„NArg i.ϊ l2-ι ^.E li To 350 ml of anhydrous THF on ice was added 3.635g (37.2 mmols) of N,0-dimethylhydroxylamine hydrochloride . The mixture was stirred for 10 minutes to allow most of the N,O-dimethylhydroxylamine hydrochloride to dissolve. Then, the following ingredients were successively added to the flask: lOg (33.8 mmols) of N-Boc-0-methyl-L- tyrosine, 6.977g (33.8 mmols) of dicyclohexylcarbo- diimide, 1.96g (16.04 mmols) of 4-dimethylaminopyridine , and 6.209ml (35.64 mmols) of N,N-diisopropylethylamine . When all of the reagents had been added, the reaction flask was placed in a cold room (4°C) and stirred for 12 hours. The contents of the flask were gravity filtered using Whatman Qualitative #1 filter paper. The filtrate was concentrated by means of a rotary evaporator to viscous oil which was then redissolved in 200 is of methylene chloride. This crude reaction mix was allowed to sit at 4 β C for one hour and then filtered as before in order to remove any residual dicyclohexylurea. The filtrate was again concentrated by means of a rotary evaporator and redissolved in 50 ml of methylene chloride in preparation for column chromatography. Column chroma¬ tography was performed using silica (230-400 mesh, 60A) as the adsorbent and 50/50 ethyl acetate/hexane as the eluant. The column used for this scale reaction was 70 cm in length and 10 cm in diameter. The product eluted after approximately 400 ml of eluant had been run through the column. The fractions were all checked by TLC using Silica Gel 60 F-254 glass backed plates. The desired product (Rf value of 0.46) was pooled and concentrated in vacuo . Concentration afforded clear, colorless oil which was placed under high vacuum for several hours . At the end of this time the product remained as a semi-solid material and with time became completely solid. There remained 5.73 g (50.2%) of a white solid with a mp of 58-62°C; IR (cm "1 , KBr) 3320, 2980, 2840, 1710, 1655, 1520, 1205, 1180; MS mle 338.4 (M+) ; X H (CDC1 300 MHz) 57.08 (d,2H, J-8.50 Hz) , 56.82 (d, 2H, J-8.50 Hz) , 55.15 (br d, IH, J-8.90 Hz), 54.89 (br m, IH) , 53.78 (s, 3h) , 53.66 (s, 3H) , 53.17 (br s, 3H) , 52.99 (d of d, IH, J-6.0 Hz), 52.83 (d of d, IH, J-6.0 Hz) , 51.39 (s, 9H) ; Anal. Calcd; C, 60.35; H, 7.69; N, 8.28. Found: C, 60.58; H, 8.02; N, 8.31. N^£^But£xγ£ rb£n l ^0^meth£xy_^L^£ r£ i.na] 1 : To 150 ml of anhydrous ethyl ether was added 1.04g (27.37) mmols of lithium aluminum hydride and the suspension was gently refluxed for 30 minutes. Upon cooling to 4°C, the reflux condenser was replaced by a pressure equalizing dropping funnel containing 7.4g (21.9 mmols) of N- (t-butoxycarbonyl) -O-methyl-L- yrosine N-methoxy-N-methylamide dissolved in 100 ml of anhydrous ethyl ether. The contents of the funnel were added over one hour. The reaction mix was allowed to react for an additional two hours. At the end of this time a cold solution of KHSO, (5.433 g in 230 ml of H 2 0) was added to the reaction vessel. The layers were separated and the aqueous layer was extracted three times with 150 mis of ether each time. The ether layers were combined and worked up as follows: washed three times with 200 mis of 3N HC1; washed three times with 200 mis of saturated sodium bicarbonate; washed three times with 200 mis of brine; and dried over magnesium sulfate, filtered , and concentrated in vacuo . There remained 3.78 g (61.6%) of a white solid with a mp of 69-72 β C: Rf-0.65 in 50/50 ethyl acetate/hexane; IR (cm " , KBr) 3360, 2840, 1740, 1695, 1530, 1255, 1180: MS m/e 279.3 (M +) ' 1 H (CDC1 3 ), 300 MHz) 59.63 (s, IH) , 57.08 (d, 2H, J-8.5 Hz) 56.84 (d, 2H, J-8.5), 55.05 (br s, IH) , 54.40 (m, IH) , 53.79 (s, 3H) , 53.06 (d, 2H, J-6.50), 51.44 (s, 9H) ; 13 C NMR (CDC1 3 , 75.47 MHz) 5200, 158.79, 130.28, 127.69, 114.27, 61.05, 55.29, 34.70, 28.26; Anal. Calcd: C, 64.51; H, 7.52; N, 5.01. Found: C.64.60, H, 7.86; N, 4.93. ^BOC :ι 4MeTy.r ΦCH ? NH2Ar£ T£s2.OH To a flask containing 100 ml of Methanol :Acetic Acid (99:1) was added 4.92g (15 mmols) of N s -Tosyl-arginine followed by 1.15g (18 mmol) of sodium cyanoborohydride. The reagents were stirred for 5 minutes followed by the addition of 4.46g of N-B0C-4-Me-Tyrosinal . After 30 minutes, an additional 1.15g (18 mmols) of sodium cyanoborohydride was added to the reaction vessel. Three additional portions of sodium cyanoborohydride were added at thirty minute intervals and the reaction was allowed to stir overnight. The reaction was worked-up by evaporating the solvent. The residue was dissolved in heptane and dried followed by dissolution in ether and drying. Water (200 ml) was added to the flask and the solid collected by filtration. TLC analysis revealed a homogenous product with an Rf of 0.35 in CHC13 :MeOH, 4 : 1) . NMR was consistent with the expected product. N^B0C^4MeTy_r ΦCH^NJ.ZliArg T£s^ :1 0H To 2.14 g (3.61 mmole) of the pseudodipeptide above was added 1.65 g (19.6 mmole) of NaHCO- in 100 ml of 1:1 dioxane/water . Benzyl chloroformate (0.6 ml, 4 mmol) was added and the reaction was stirred overnight. The solvents were removed in vacuo leaving a gummy residue. The residue was suspended in 100 ml of water and this was acidified to ρH2 with HC1 and extracted three times with ethyl acetate. The combined ethyl acetate fractions were dried over magnesium sulfate, filtered and evaporated to provide 2.35g (90%) of the desired material as a crude amorphous white solid. Recrystallization from methylene chloride/hexane provided 2.18g (83%) of the product as a white solid. ^^-tS£llS£2£_£-f_-£^£_-∑£2££-t£ _S H5£dl.E£Etide_t£_the Z£.-kϊ.£_t∑. r . 2 _--- .--.i2 The protected pseudodipeptide was attached to hydroxymethyl resin (Polystyrene-1% Divinylbenzene , 0.7 mequiv./g) using dicyclohexylcarbodiimide and 4-dimethyl- aminopyridine . To 1.87 g of hydroxymethyl resin (1.31 mmol) was added 2.28 g (3.28 mmol) of the protected pseudodipeptide, 410 mg (3.33 mmol) of 4-dimethylamino- pyridine, and 25 ml of anhydrous dimethylformamide in a 50 ml polypropylene tube. To this was added 680 mg (3.3 mmol) of dicyclohexylcarbodiimide and the vessel was shaken overnight at room temperature. The resin was collected by filtration and washed successively three times each with methylene chloride and methanol and dried overnight in vacuo to provide 2.6g of resin. Sub¬ stitution by weight gain was calculated to be 0.54 mmol/g . Exam£ ] 1 e_I ^ V_ : _ .Sχ2-£!l£ i _ 2£^--_Hϊi-£l£ -£i 2_£.f-_Δ- l Z A-7 was prepared by solid-phase peptide synthesis by sequentially assembling the remaining amino acids (in a C- to N-fashion) onto the resin bound protected pseudo¬ dipeptide. The peptide was synthesized on a Beckman 990 peptide synthesizer using the following program for each coupling cycle, 1- Wash, CH„C1„ (3 x 1 min); 2-Deprotect 40% TFA/CH 2 C1 2 (2 x 10 minutes); 3- Wash, CH 2 C1 2 (3 x 1 min); 4 - Wash. Isopropanol (2 x 1 min) ; 5 - Wash, CH„C1 (3 1 min); 6- Neutralize 5% DIEA/CH 2 C1 2 (3 2 min) ; 7 - Wash, CH 2 C1 2 (5 x 1 min); 8 - Couple (3 equivalents Boc-Amino acid, 3 equivalents of BOP) 1 x 60 min; 9 - Wash, CH„C1„ (3 x 1 min); 10 - Wash, Isopropanol (2 x 1 min); 11 - Wash, CH 2 C1 2 (3 x 1 min); 12- Test for coupling by ninhydrin. If recoupling was necessary as judged by a positive ninhydrin test, a partial cycle starting at step 6 to the end was done. Following assembly of the complete protected peptide, the N-terminal BOC group was removed by using steps 1-5 in the cycle and the resin dried. The crude peptide was isolated from the resin by treatment of the protected peptide-resin with anhydrous HF containing 10% anisole for 1 hour at 0 β C. The HF was removed in vacuo and the crude resin/peptide was washed with ether three times . The peptide was extracted with 10% acetic acid and lyophilized to yield a crude peptide. The peptide was partially purified by HPLC using a 0.1% TFA/acetonitrile gradient (10-40% over 30 minutes) on a C - a reverse phase support. The peptide was further purified using isocratic 15% acetonitrile in 0.1% TFA as an eluant on a C. „ support. The fractions from the main peak were combined to provide purified A-7 which appeared homogeneous by TLC, Electrophoresis , and HPLC. FAB/MS analysis yielded the expected MW of 1098. Amino acid analysis often 6N HC1 hydrolysis (24 h @ 110°C) gave the following composition: (Ser(l)0.89, Pro (2)2.00, Gly (1)0.97, Arg (1)1.03, Thi(l)0.73g 4Me-Tyr- ( CH 2 NK) - Arg(l) , detected by an alternate method, was partially destroyed in hydrolysis. Exam£ ] .e_V_ : Tim _C_£ur££_£f_B]. £d_ Brai.n_Barri. r_θ i_ng_i. Mi£ _and_Rat . Female Balb/C mice weighing approximately 20 g were use. All solutions were prepared in sterile phosphate- buffered saline. Intravenous injections of 100 μl of the 3 5 bradykinin analogue and agonist [Hyp , Thi ,4-Me- 8 9 Tyr Φ(CH_NH)Arg ]bradykinin (hereinafter designated as A-7) were given in the tail vein at time - 0. This agonist differs from bradykinin in that the 3rd (Pro) amino acid has been replaced by hydroxyproline , the 5th (Phe) amino acid has been replaced by thienylalanine, the 8th (Tyr) amino acid has been substituted with a methyl group at the 4 position and the peptide bond between the 8th (4-Me-Tyr) and 9th (Arg) amino acids is replaced with the isosteric CH--NH bond. At various specified times following the injection of A-7 an injection of 14 6 C-sucrose (3 X 10 dpm) was also made in the tail vein. Two, five or ten minutes later, the mice were killed. Blood was collected in heparinized tubes and centrifuged to separate plasma from the resulting pellet. A 100 μl aliquot of plasma was counted by liquid scintillation counting after the addition of 15 ml of Aquasol-2 (Dupont) . The brain was removed and homogenized in 2.5 ml of water and 2.5 ml of 1% SDS. One ml of the homogenate was aliquoted and added to 15 ml of Aquasol-2 for counting. Brain uptake of sucrose was calculated and expressed as percent of injected dose X 100. The results are displayed in Figure 2. As seen in 14 this Figure, the uptake of C-sucrose at 10 and 20 minutes was significantly higher in the presence of A-7. The blood-brain barrier remained permeable to sucrose for at least 20 minutes following injection of A-7 but did not remain open after 40 minutes. Each data point repre¬ sents the mean ± s.d. from 8 mice. The time course of blood-brain barrier opening in rats is shown in Figure 3. The methodology is essentially the same as in the above description except that female Harlan Sprague-Dawley rats (150-200 g) were 6 14 used and 6 X 10 dpm of C-sucrose were used. The blood-brain barrier remains open to sucrose for 20 minutes in rats following A-7 injection. Each data point represents the mean ± s.d. from 6 rats. In some experiments whole blood samples and intact brain tissue were combusted in a Packard Model 307 Oxidizer and the radioactivity collected and counted in a liquid scintillation counter. The results obtained by either homogenization or combustion of samples were equivalent . Exam ^ _V]._ : D£ _R £n£e_R l ^ a£i : £ hi ; _£f_Bradγki ^ ^n_and The methodology for these experiments is similar to the above two time course studies with the exception that all mice were killed 10 minutes after receiving a single 14 tail vein injection of C-sucrose simultaneously with bradykinin (BK) or bradykinin analogue. Figure 4a shows 14 the amount of C-sucrose uptake expressed as 100 X percent of injected dose over various concentrations of bradykinin andbrandykinin analogues after 10 minutes. Figure 4b represents the percent maximum response (sucrose uptake) over the range of doses of bradykinin or bradykinin analogue shown. The bradykinin analogue and agonist ( [Thi 5 , Phe 8 (CH 2 -NH) Arg 9 ]bradykinin) (A-4) and, particularly, [Hyp ,The , 4-Me-Tyr Φ(CH 2 NH)Arg 9 ]bradykinin (A-7) are more potent than the combination of bradykinin and captopril (BK+Cap). The number of mice per data point ranged from 12 to 16. Exam ^ _v:[,I_. U£take_£f_ ub £an££s_£f_Di.fferent_Molec l r Wϊi- j gh _^n££_£he_Bra^n_£f_Mi^ The methodology of these experiments is similar to that of the above two examples. Specific radioactively labeled molecules of different molecular weight and structure, either with saline or with 10 μg of A-7, were intravenously injected into mice via the tail vein. The animals were sacrificed 10 minutes after the coinjection and the radioactivity present in brain was measured as 14 described previously for C sucrose. The results of these studies are shown in Table I. TABLE_I U££ake_£f_Rad^£] ; ab l ^ l ^ d_ ub £an£ _^nt£_th _Brai ^ n i ££i U2.λ^ l E-l t ££2£-££l ±_i^H£_ ^Z_ 14 C-Sucrose 342 5.9±1.4 16.5±4.9 H-Inulin 5,000 0.075±0.018 O.HO±O.016 3 H-RNase 12,600 0.12310.039 0.117±0.030 H-Myoglobin 14,000 0.092+0.022 0.07310.014 3 H-Carbonic Anhydrase 30,000 0.09010.013 0.10610.015 3 H-Ovalbumin 46,000 0.07910.016 0.09310.017 3 H-Bovine Serum Albumin 68,000 0.33310.098 0.20910.056 Data are presented as mean 1 s.d. for 7 to 15 mice in each group. It appears that substances ' with greater molecular weights do not readily cross the blood-brain barrier when coinjected with A-7. This suggests that the blood-brain barrier permeabilizer characteristics of A-7 are restricted to lower molecular weight substances. --------t---£-k≤._---il.l.--_Σli£_ ^-t££-£_£-t_- -tH-il -tit.i2iB_^2£ , _A2 i££H _£ the_Brain_U£take_and_Ant^n££i£e_£ti.V£_Effec£_£f i-£E£---- 5 ^£-^ ϊ≤i1_Fl^£ _A ay_• --i.--------lZ_---i. B-ι: ( ϊ∑-k:-i-2i2 Female Balb/C mice weighing approximately 20g were used. The tail flick assay was performed using a Tail Flick Apparatus model TF6 (Emdie Instruments, Maidens, VA) . The intensity of the radiant heat was adjusted daily to yield a reaction time between 2 and 3.5 seconds in naive untreated mice. The cutoff time was set at 7.5 seconds. The tail withdrawal reaction time of mice was measured three times at 10 second intervals immediately prior to intravenous injections. These three values were averaged and taken as basal value (V ) . Another three measurements were taken 10 minutes after intravenous injections and averaged ( V ι0 ) • * n some experiments, the opiate receptor antagonist naloxone (NAL) was adminis¬ tered intraperitoneally 15 minutes (100 μl in sterile PBS, 10 mg/kg) prior to intravenous administration of bradykinin and loperamide (BK+LOP) . The results were expressed as percent antinociceptive response according to the formula: 100 x V 1Q - V Q )/(7.5 - V Q ) . Figure 5 illustrates the effectiveness of bradykinin on the brain uptake of the opiate receptor agonist loperamide (LOP) by demonstration of antinociceptive effects. While loperamide injected intravenously at a dose of 25 μg per mouse had no activity, a complete antinociceptive response was observed when bradykinin (30 μg) was co-injected with the opiate, with the latency of tail withdrawal reaching the cut-off limit of 7.5 seconds in all mice. Pretreatment of mice with naloxone (10 mg/kg) completely antagonized this antinociceptive activity . Study__B _ IHy_ J _Thi^4 jL Me^T .r^Φ CH„NH^A^^ Female Balb/C mice weighing approximately 20 g were used. The tail flick assay was performed using a Tail Flick Apparatus model TF6 (Emdie Instruments, Maidens, VA) . The intensity of the heat source was adjusted daily to yield a reaction time between 2.0 and 3.2 seconds in naive untreated mice. The maximum time of exposure to the heat source allowed was 7.5 seconds. The tail withdrawal reaction time of mice was measured 4 times at 10 second intervals immediately prior to intravenous injections via the tail vein. The last three values were averaged and taken as the baseline value (V-) . Another set of measurements was taken at the following intervals after tail vein injection: immediately, 5 min, 10 min, 15 min, 30 min, and 60 min. The last three values for each of these time points (V) was averaged. In some experiments the opiate receptor antagonist naloxone (10 mg/kg; 100 μl in saline) was administered intra- peritoneally 15 minutes prior to administration of A-7 (0.1 μg) and loperamide (25 μg) . The results were expressed as percent antinociceptive response according to the formula: 100 X (V-V Q )/(7.5-V ) . Figure 6 illustrates the ability of A-7 to enhance, the permeability of the blood-brain barrier to loperamide as evidenced by the increase in % antinociceptive response. Each point represents pooled data from 2 experiments with 4 mice (total of 8 mice) 30 minutes after injection of loperamide, A-7, and A-7 and loperamide with or without naloxone pretreatment. A complete antinociceptive response was obtained when A-7 was coinjected with loperamide. The effect was com¬ pletely antagonized by pretreatment with naloxone. i. § £_ε∑if!i£E m j Ln _Wh n_C£^Adm^ni £ r d_wi^h ^ £ii£_-t£ £S£££Σ_ ££iZUΣ_£-f_S≤t . Five Sprague-Dawley rats (125-150g) were habituated to activity cages two days prior to the experiment. The activity cages were standard size rat cages with two photocell beams running across the floor of each cage. Any movement of the rat through either of the beams was recorded by a computer. Locomotor activity was measured as the number of beam breaks in a sequence of 10-minute intervals over two hours. On test days, all rats were pretreated with the monoa ine oxidase inhibitor nialamide (100 mg/kg i.p.). This was done to prevent the enzymatic degradation of diacetyldopamine (AcDA) by monoamine oxidases. They were then placed in the activity cages to habituate. Two hours later, rats were briefly removed from the cages and injected intravenously via tail vein injection with either AcDA (2mg) or AcDA (2mg) + bradykinin (BK) (l g) . The rats were immediately returned to the activity cages and locomotor activity was recorded over 10 minute intervals for the next two hours. The results are displayed in Figure 7. As this Figure shows , those rats receiving AcDA alone showed no increase in locomotor activity during the two hour test session. However, when AcDA + bradykinin were co- injected, there was a significant increase in the loco¬ motor behavior of the rats. These data suggest that bradykinin had enabled AcDA to enter the brain resulting in stimulation of central nervous system dopamine receptors . In order to verify that the motor increase seen was due to direct stimulation of dopamine receptors , one group of rats received the dopamine antagonist halo- peridol in addition to AcDA + bradykinin. The halo- peridol (0.2 mg/kg i.p.) was administered 15 minutes prior to the AcDA/bradykinin injection. As can be seen in the figure, the increased motor activity was com¬ pletely abolished with the haloperidol pretreatment. Exam£l L £_X_ : _h _Eff £_£f_ _D£ ami.n rgi _An a££ni £_Wh ^£^adm^ni £ red_w^£h_A^ _£2_£-tl _i; ££5!££££_A££i.vf.t2_£f Rats. Domperidone is a dopamine receptor antagonist used clinically as an anti-emetic by virtue of its action at the area postrema outside the blood-brain barrier. Reports in the literature demonstrated that domperidone does not cross the blood-brain barrier but when given as an injection into the cerebral ventricles, it effectively blocks binding of dopaminergic compounds, such as apomor- phine , to dopamine receptors. A pertinent test is whether domperidone can antagonize a dopamine receptor agonist- induced increase in motor activity when co- administered with A-7 but is ineffective when administered without A-7. Sprague-Dawley rats (125-150g) were habituated for two days to activity cages. The activity cages were standard size rat cages with two photocell beams running across the floor of each cage. Any movement of the rat through either of the beams was recorded by a computer. Locomotor activity was measured as the number of beam breaks in a sequence of 10-minute intervals over a two hour period. The rats were given a coinjection of lOμg A-7 and 300μg domperidone, or the domperidone alone, one hour before a subcutaneous injection of apomorphine (0.75mg/kg) , which is a dopamine agonist. The motor activity of the rats was measured in activity cages over 10-minute intervals for up to 2 hours post-apomorphine inj ection. The results of this experiment with 3 rats in each treatment group are shown in Figure 8. The combination of A-7 and domperidone antagonized the increase in motor activity associated with apomorphine. Domperidone alone had little, if any, effect on motor activity induced by apomorphine which readily crosses the blood-brain barrier . E m£jL _XI^ ∑he_Effe£t_£f_the_Anti_ Tum£r_Ag nt 2i £i5^i.2_5ll 2_5£ .^Si2i -t -£ ^---.ϊi£^-_- Σ^^ϊli i2_ 2^ £a££££riJL_£r_a_Brady_ki.nin_AnaJ L ££££_£2_£ii£_ 2ΣXiZ£i_ϊi2 £f_Rat _wi.£h_Brai.n_Tum£r_I ^ m£j.ant . Male Fisher 344 rats (200-250g) were anesthetized with ketamine HC1 (100 mg/kg) and acepro azine (10 mg/kg). The animals were placed in a stereotaxic frame. The heads of the animals were shaven and a midline incision was made to expose the skull. A small hole was drilled over the right sensorimotor cortex. Lastly, a 100 μl cell suspension (9L glioma cells, 2.5 x 10 cells/ml) was injected over 5 minutes into the right caudate putamen of each animal and the scalp sutured. On the days that followed, the animals received treatments as described below. From day 4 until the end of the study or time of death, animals were observed daily for signs of failing health. When signs of very poor health were observed (eye hemorrhage, loss of righting reflex) , animals were killed and perfused with paraformaldehyde for subsequent histology to verify the existence of the tumor . j3tudy__A Rats were treated on days 4 and 6 after implantation of the tumor with 600 μg cisplatin i.v. via the tail vein (18 rats, Cis) , or 1 mg of captopril i.p. followed 15 minutes later by 1 mg bradykinin i.v. (18 rats, Cap-Bk), or 1 mg captopril i.p. followed 15 minutes later by 1 mg bradykinin i.v. followed immediately thereafter by 600 μg cisplatin i.v. , (18 rats, Cis/cap-bk). Another group of rats received no treatment (17 rats, control) . The results are shown in Figure 9, where survival time versus number of live rats is plotted. Treatment with captopril plus bradykinin had no effect on survival time. Treatment with the antineoplastic agent, cisplatin, did however significantly increase survival time and pretreatment with captopril and bradykinin before administering cisplatin significantly augmented this effect. The curves were fitted assuming they follow a Weibull distribution. The mean survival times and corresponding standard deviations are given in the following table in days: TABLE_II G_££U£ *£ n -§.-__ . 0. Control 10.0 2.37 Cisplatin 13.1 2.90 Captopril-Bradykinin 10.0 1.84 Cisplatin/captopril-Bradykinin 16.5 5.77 When the anti-tumor agent, cisplatin, was administered alone, the survival time increased from 10.0 to 13.1 days. However, when cisplatin was co-administered with bradykinin and captopril, the survival time increased even further to 16.5 days. These data suggest that the pretreatment of bradykinin (with captopril to prevent its degradation) increases the blood-brain barrier's perme¬ ability to cisplatin and thereby increases the anti-tumor agent's effectiveness in the brain. The Cis/Cap-Bk group was statistically significantly different from the Cis group with a p value less than 0.025. tudγ_B Rats were treated daily from day 4 to day 14 after implantation of the tumor with 200 μg cisplatin i.v. (9 rats, Cis) or 1 mg captopril i.p. followed 15 minutes later by 1 mg bradykinin i.v. plus 200 μg cisplatin i.v. (10 rats, Cis/Cap-Bk). Another group received captopril and bradykinin, without cisplatin (9 rats, control). The results are shown in Figure 10 and confirm the earlier results in Study A which demonstrated that pretreatment with bradykinin and captopril significantly increase the effect of cisplatin on the survival time of rats with brain tumor implants. The mean survivals and corresponding standard deviations are given below in days : TABLE_^II §££££ Mean ± L-D^ Control 8.1 2.05 Cisplatin 11.9 2.62 Cisplatin/Captopril-Bradykinin 19.7 2.45 The survival times of rats receiving cisplatin with bradykinin and captopril were significantly higher than rats receiving only cisplatin. The Cis/Cap-Bk group was statistically different from the Cis group with a p value less than 0.005. The increased permeability of the blood-brain barrier caused by the administration of bradykinin (with captopril to prevent its degradation) , apparently increased the brain uptake of cisplatin and thereby increased its therapeutic effectiveness. £tudy__C j _ IHy.£ 3 _^Thi£^4^Me^Tχr^ CH NH2Ar£^lbr dy_ inin Male Fisher 344 rats (200-250 g) were anesthetized with ketamine HCL (100 mg/kg) and acepromazine (10 mg/kg) . The animals were placed in a stereotaxic device. The heads of the animals were shaved and a midline incision was made to expose the skull. A small hole was drilled over the right sensorimotor cortex. A 100 μl cells suspension (250,000 9L Glioma cells) was injected over 5 minutes into the right caudate putamen of each animal and the scalp sutured. Animals were observed daily for signs of failing health. When signs of very poor health were observed (eye hemorrhage or loss of righting reflex) animals were killed and the brains examined for presence of tumor. On days 5 through 14, the animals received the following tail vein intravenous treatments: no treat¬ ment; cisplatin 200 μg/rat; A-7 (50 μg) and 5 minutes later cisplatin; or captopril pretreatment followed by 1 mg bradykinin 15 minutes later and cisplatin 5 minutes after bradykinin. The results are shown in Table IV as median with range. TABLE IV Treatm nt_Gr£U£ M d^an_ urvival__ da ; •5£^._Animals Control 14, Range 10-16 6 Cisplatin 13, Range 9-18 9 BK & Captopril + Cisplatin 16, Range 10-21 9 A-7 + Cisplatin 20.5, Range 10-62 9 Figure 11 illustrates the survival times of all animals in the study. It should be noted that 2 animals in the A-7 + cisplatin treatment group had extended survival times, with one animal dying on day 38 and the other sacrificed at day 62. Both animals had evidence of tumor growth. Exam£l.e_XI[ Eff£££_£f_Bradγkinin_£n_th _Brai.n De].i.V rγ_£f_Vari.£U _M£^e£u].e The protocol for these experiments was the same as described in Example I, except as noted. Radiolabeled compounds (i.e. , molecules) were administered intra¬ venously to the mice with or without 300 μg of bradykinin. Brain levels were determined 10 minutes following treatment. The data are derived from 2-3 independent experiments for each compound. Four mice were used for each different molecule per experiment. The results were as follows: Table_V ---£i£££i------_---t-.-- i2 -E£ e_i.n Z Σ£ 2£_ . ££ £-f_5£2-UΣ i 77 + 24%* 3 H-AZT 267 +6.5 1 2.6* 34 + 10%* 14 C-N-acetyl- Amphotericin B 1,000 +2.9 1 0.9* 24 1 7%* 14 C-CD-4 40,000 +1.9 1 1.3 19 + 13% * Statistically significantly higher than brain uptake without bradykinin. Bradykinin significantly increased the brain uptake of sucrose, AZT and N-acetyl-Amphotericin B. At a molecular weight of 40,000, however, the uptake of CD-4 was not significantly affected. The data indicate that bradykinin preferentially increases the blood-brain barrier permeability to small molecular weight substances . Exam£le_XIII L ^™Ϊ ι2ISIDA_ N^χ2_ L ^^Diis£ r£ γl^ a tan^l^dej_^mϊn£d^acet^£_a ^d _U tak _^nt£_th _H ad £Ϊ££_ Brai£i_i _--------:£• Female Sprague-Dawley rats (250-300g) were anes¬ thetized with pentobarbital (60 mg/kg) and ketamine (100 mg/kg) . The femoral veins were surgically exposed and the right vein was injected either with saline or a range of A-7 concentrations. After three minutes, a bolus of Tc-DISIDA was injected into the left femoral vein. The rats were immediately placed on a gamma camera and the radioactivity counted intitially at 1 minute intervals and then at 5 minute intervals for 1 hour. The head region where the brain is the primary organ was identified and the amount of radioactivity present in this region is shown in Figure 12 for each of the con¬ centrations of A-7 tested. The data for each con¬ centration are radioactivity measurements from a single rat. At very early times the A-7 enhanced the uptake of Tc-DISIDA into this region relative to the control animal. This experiment is representative of two similar studies . In another set of experiments, a single intravenous injection of A-7 was given into a femoral vein of an anesthetized rat. Two minutes later, an injection of 9 m Tc-DISIDA was given into the contralateral femoral vein. In control animals, no A-7 was injected (sham injection). At time intervals of 2, 10, 30, or 60 99m minutes after the Tc-DISIDA injection, the rats were sacrificed, their brains removed and counted in a gamma counter. The brain uptake of Tc-DISIDA was calculated and expressed as percent of injected dose per organ. The biodistribution of Tc-DISIDA in the whole brain of untreated and A-7 treated rats at selected times post- injection is shown in Table VI and Figure 13. TABLE_VI. Biodistribution of 99m Tc-DISIDA in Brain Time % Injected Dost/Brain 99m [after Ϊ£ .lHIDA_^ 2ecti£n C£ntr£3. +_10 £_A_ 2 2 min. 0.04010.013 0.07510.019 5 min. 0.03210.003 0.04610.006 10 min. 0.02210.005 0.02810.003 60 min. 0.00410.001 0.01010.003 The data are expressed as mean 1 s.d. for three animals per group . These results demonstrate that larger amounts of 99m Tc-DISIDA are found in the brain of A-7 treated rats when compared to control rats at early times post- injected of the labeled agent. Exam£le_XIV_ : _ The_Effe£t_£f_An£i.£ten in_iI_When_C£^ S^Si2i -ζ£Σ£^_^i : -£^_ i .Z_ 2_--ϊ £---.M∑i li2β_-§£ ≤∑i££_£f_ t . Angiotensin II in supra-physiological concentrations has been shown to induce drinking behavior in water satiated rats. This behavior has been suggested to occur as a result of stimulation of angiotensin II receptors within areas of the brain not associated with the cerebroventricular organs. Studies were performed to evaluate the effect of co-administration of A-7 with an angiotensin II analogue that is capable of causing drinking behavior when administered at a high dose. Rats were given a coinjection of lOμg A-7 and either 0.1, 0.3, 3, 10 or 30μg of 3-angiotensin II, or the )3-angiotensin II, alone, via tail vein injection. The volume of water consumed by each rat over a 1 hour interval was measured. The results of this study with 6 rats in each dosage group are shown in Figure 14. Co-administration of A-7 and the angiotensin II analogue caused the dose response curve to shift to the left, or toward lower doses of analogue, when compared to administration of the analogue alone . In another study, rats were given either saline, 1 μg of ^-angiotensin II, 1 μg of ^-angiotensin II and 10 μg of A-7, lμg of β -angiotensin II and 8 μg of saralasin or 1 μg pf ^-angiotensin II plus 10 μg of A-7 plus 8 μg of saralasin via tail vein injection. The saralasin was given because it is a known angiotensin II receptor antagonist. Again, the volume of water consumed by each rat over a 1 hour interval was measured. The results of this study with 3 rats in each group are shown in Figure 15. The co-administration of A-7 and β-angiotensin II causes a significant increase in water consumption compared to the angiotensin II analogue alone or together with saralasin. When saralasin is co-administered with A-7 and the analogue, the water consumption remains within a normal range which indicates an inhibition of angiotensin II analogue-induced drinking behavior by the addition of the angiotensin II receptor antagonist .

Claims: CLAIMS 1. A method for increasing the permeability of the blood-brain barrier of a host to a molecule present in the blood stream of the host comprising co- administering intravenously to said host an effective amount of a bradykinin agonist of blood-brain barrier permeability. 2. A method of Claim 1 wherein the host is a human being. 3. A method of Claim 2 wherein the bradykinin agonist of blood-brain barrier permeability comprises bradykinin. 4. A method of Claim 2 wherein the bradykinin agonist of blood-brain barrier permeability comprises a bradykinin analogue. 5. A method of Claim 4 wherein said bradykinin analogue has significantly increased resistance to proteolytic degradation in the blood stream of the human being relative to bradykinin. 6. A method of Claim 2 wherein the bradykinin agonist of blood-brain barrier permeability comprises compounds that mimic the increase in permeability of the blood-brain barrier in the manner of bradykinin. 7. A method of Claim 1 wherein the bradykinin agonist of blood-brain barrier permeability is selective for bradykinin 2 (B_) receptors. 8. A method of Claim 2 wherein the bradykinin agonist of blood-brain barrier permeability and said molecule are simultaneously intravenously administered to the host. 9. A method of Claim 1 wherein the molecule comprises a diagnostic imaging agent. 10. A method of Claim 9 wherein the diagnostic imaging agent is radiolabelled or doped with a magnetic resonance imaging contrast substance. 11. A method of increasing the permeability of the blood-brain barrier of a host to a molecule present in the blood stream of the host comprising co- administering intravenously to said host an effective amount of a blood-brain barrier permeabilizer comprising a receptor mediated permeabilizer. 12. A method for increasing the permeability of the blood-brain barrier of a host to a neuropharma- ceutical agent present in the bloodstream of the host comprising co-administering intravenously to said host an effective amount of a bradykinin agonist of blood-brain barrier permeability. 13. A method of Claim 12 wherein the bradykinin agonist of blood-brain barrier permeability and said neuro- pharmaceutical agent are simultaneously and intravenously administered to the host. 14. A method of Claim 12 wherein said neuropharma- ceutical agent is cisplatin. 15. A method of Claim 12 wherein said neuropharma- ceutical agent is amphatericin B. 16. A method of Claim 12 wherein said neuropharma- ceutical agent is azidothymidine . 17. A method of Claim 12 wherein said neuropharma- ceutical agent is clindamycin. 18. A method of Claim 12 wherein said neuropharma- ceutical agent is gentamicin. 19. A method for increasing the permeability of the blood-brain barrier of a human to a therapeutic agent present in the blood stream of said human comprising intravenous co-administration to said host of an effective amount of a bradykinin agonist of blood-brain barrier permeability. 20. A method of treating a patient afflicted with toxoplasmosis encephalitis by administering to said patient a therapeutically effective amount of an anti-parasitic agent and co-administering therewith a bradykinin agonist of blood-brain barrier permeability. 21. A method of Claim 20 wherein said anti-parasitic agent comprises gentamicin. 22. A method of Claim 20 wherein said anti -parasitic agent comprises clindamycin. 23. A pharmaceutical composition for intravenous administration to a human for the purpose of increasing the permeability of the blood-brain barrier to a molecule, comprising: a) the molecule to be delivered from the blood to the brain; b) a bradykinin agonist of the blood-brain barrier permeability; and c) a pharmaceutically acceptable carrier.

Inventors: MALFROY-CAMINE BERNARD, SMART JANET L, MALFROY-CAMINE, BERNARD, SMART, JANET, L.
Applicants: ALKERMES INC, ALKERMES, INC.
Application year: 1991
Main classification: C07K99
IPC codes: A61K31, A61K33, A61K38, A61K47, A61K49, A61K51, A61P25, A61P43, C07K7