- Email: [email protected]
[29] Targeting of drugs to the brain
[29]
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These results indicate that prodrugs targeted for intestinal peptidases as well as those targeted for intestinal alkaline phosphatase show potential for improving oral drug delivery. For the stable phosphate derivatives, axial enzyme distribution in different species should determine the relative success for these prodrugs. For those prodrugs targeted for intestinal peptidases, prodrug instability and enzyme specificity with respect to targeting to gain transport advantages for oral administration are key considerations for the utility of this approach. Acknowledgments The authors acknowledge the support of NIH Grant No. 1ROIGM3169 for much of the work presented in the Results section and thank Iris Templin for her expert assistance in preparation of the chapter.
[29] T a r g e t i n g o f D r u g s to t h e B r a i n By NICHOLAS B o o o a Introduction The overall membrane transport properties are very important for a drug, as these properties govern the absorption, distribution, and elimination of its intact form, and affect binding, affinity, and other important characteristics. It is of even greater importance to find methods which enable delivery of drugs specifically to a particular organ, or site. This requires more than simply optimizing overall membrane transport characteristics. Among the various possible ways to achieve site-specific or organ-specific delivery, the chemical delivery system (CDS) is the most flexible)--and it offers possibilities for specific delivery not only to the brain, or eye 2 but to other organs and sites. Properly designed, a CDS or any other drug delivery system should concentrate the desired active agent at its site of action and reduce its concentration in other locations. The main result of this manipulation is not only an increase in the efficacy of the drug entity but also a decrease in its toxicity. A site-specific delivery designed for the central nervous system (CNS) would be especially useful. It is well known that many of the pharmacologically active and t N. Bodor and H. H. Farag, J. Med. Chem. 25, 313 (1983). 2 N. Bodor and G. Visor, Exp. Eye Res. 38, 621 (1984).
METHODS IN ENZYMOLOGY,VOL. 112
Copyright © 1985by Academic Press, Inc. All rightsof reproductionin any form reserved. 1SBN 0-12-182012-2
382
PRODRUGS
[29]
important agents, including endogenous neurotransmitters, cannot be transported from the blood into the brain because of a set of specialized barriers present at the blood-brain interface. This barrier system is generally called the blood-brain barrier (BBB) and is composed of a set of anatomical and enzymatic components. The existence of a barrier which separates the general circulation from the central nervous system was first postulated by Ehrlich at the end of the nineteenth century. 3-5 The actual barrier was later described as acting against complexes formed between the dyes and plasma proteins with which these compounds are extensively bound. However, it was discovered that many small compounds are also unable to pass this barrier. The morphological basis of the BBB was for long a rather controversial issue. Most recently, the anatomical basis for the BBB was identified as the endothelial lining of the cerebral capillaries, and most importantly, not the perivascular cells. There are several ultrastructural differences between systemic capillaries and cerebral capillaries which explain the difference in their permeabilities. The main difference is in the manner in which endothelial cells in cerebral capillaries are joined. Cerebral junctions are characterized as tight or closed junctions which gird the cell circumferentially, forming zona occludens and providing an absolute barrier. Structurally, the junctions consist of aligned intramembranous ridges and grooves which are in close apposition. 6 Systemic capillaries lack this closed junction. Morphologically, this can be traced to a lack of continuity in the intercellular appositions. Materials pass easily between these leaky cells, while in the brain the sealing of the intercellular fissures severely restricts this nonspecific transport. Since intercellular transport is impossible, only intracellular or transcellular transport remains. Lipophilic compounds can readily pass through these phospholipoidal membranes, but hydrophilic compounds and high-molecular-weight substances are excluded. A second main difference between cerebral and systemic capillaries is the paucity of vesicles and vesicular transport in the CNS.7 A third difference is the lack of fenestrae in the cerebral capillaries. Cerebral vessels have a number of perivascular accessory structures which appear to be involved in the function of the BBB. Astrocytic foot processes may be involved in the regulation of the amino acid flux. Phago3 W. M. Pardridge, J. D. Conner, and I. L. Crawford, CRC Crit. Rev. Toxicol. 3, 159 (1975). 4 E. Levin, Exp. Eye. Res. 25, Suppl., 191 (1977). 5 B. Van Deurs, Int. Rev. Cytol. 65, 117 (1980). M. W. Brightman and T. S. Reese, J. Cell Biol. 40, 648 (1969). 7 M. W. Brightman, Exp. Eye Res. 25, Suppl., 1 (1977).
[29]
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cytic pericytes which are present abluminally may play a similar role. It is possible that the basement membrane of endothelial capillaries acts as a mass filter, preventing large molecules from penetrating it. In addition to these structural features, the BBB maintains a number of enzymes which appear to augment barrier function. 4,8.9 Optimal neuronal control requires a careful balancing between neurotransmitter release, metabolism, and uptake, as it is of vital importance to restrict the entry of blood-borne neurotransmitters into the CNS. This is why high concentrations of such enzymes as catechol O-methyltransfcrase (COMT), monoamine oxidase (MAO), 3;-aminobutyric acid aminotransferase (GABA-T), and aromatic-L-amino-acid decarboxylase (DOPA-decarboxylase) are found in the BBB. The enzymatic BBB may also play a role in the exclusion of some lipophilic compounds which otherwise might passively diffuse through the barrier. The transport of nutrients is brought about by a number of carriers which are situated in the endothelial cells and which are assumed to be proteinaceous materials. They are equilibrative, i.e., nonenergy dependent and bidirectional in nature, and can be saturated, m.jl The net movement of compounds is always along a concentration gradient, and since nutrients are readily utilized as soon as they pass into the brain, this gradient is in the direction of the brain. The BBB, therefore, consists of a relatively impermeable membrane superimposed on which are mechanisms for allowing the entrance of essential nutrients and the exit of metabolic wastes. If a compound is to gain access to brain parenchyma, it may do so via several routes. If the molecule has affinity for one of the carriers previously described, it may diffuse across the BBB by association with this carrier. A compound with high intrinsic lipophilicity can diffuse passively through the phospholipid cell membrane matrix. These two avenues, namely, passive diffusion and carrier mediation, represent the major components for influx. Other minor mechanisms may also allow the entry of substrates to the C N S . 7']2 Retrograde axoplasmic transport has been observed in such areas as the nucleus ambiguous and the abducens nucleus. There are several areas in the brain which lack a BBB. 13,~4 These J. E. Hardebo and B. Nilsson, Acta Physiol. Stand. 107, 153 (1979). 9 j. E. Bardebo and C. O w m a n , Ann. Neurol. 8, 1 (1980). l0 W. M. Pardridge, Diabetologia 20, 246 (1981). " W. M. Pardridge and W. H. Oldendor, J. Neurochem. 28, 5 (1977). ~z E. Westergaard, Adv. Neurol. 28, 55 (1980). 13 C. W. Wilson and B. B. Brodie, J. Pharmacol. Exp. Ther. 133, 332 (1961). t4 G. D. Pappas, J. Neurol. Sci. 10, 241 (1970).
384
PRODRUGS
[291
include locations near the ventricles such as the area postrema, the subfornical organ, the median eminence of the neurohypophysis, the organum vasculosum of the lamina terminalis, and the choroid plexus. Collectively, these areas are termed the circumventricular organ. In addition, the pineal gland also lacks a BBB. These areas constitute a small fraction of the total surface area of the BBB and may allow a limited nonspecific flux. Cerebral spinal fluid (CSF) is produced at the choroid plexus and drains from the ventricles through the foramina of Magendie and Luschka into the subarachnoid space of the brain, j5 Cerebral spinal fluid, along with any dissolved materials, leaves the subaraehnoid space via the arachnoid villi, which protrude into a venous sinus. The arachnoid villi act as a one-way valve and prevent backflow. 16 This loss of CSF provides a slow mechanism for nonspecific efflux of compounds from the CNS. The mechanism rids the brain of polar compounds such as metabolic wastes at a fairly constant rate regardless of the molecular size. Therefore, while lipophilicity is very important for influx to the brain, the efflux of a compound is only partially dependent on this parameter. 17-19 The BBB excludes a number of pharmacologically active agents and, as such, treatment of many cerebral diseases is severely limited. To increase the effectiveness of drugs which are active against these diseases, the specific transit time of a desired drug in the brain must be increased. This should increase the therapeutic index of an agent since not only is the concentration and/or the residence time of the agent increased in the vicinity of the bioreceptor, but of equal importance, the peripheral concentration of the drug is reduced, thereby decreasing any associated toxicity. Unfortunately, there are very few methods for circumventing the BBB, and these are of limited usefulness. The direct administration of drugs in the CNS, i.e., an intrathecal injection (i.t.), has been used to deliver various pharmacologically active agents. While i.t. injections are of potential value, they are notorious for their high incidence of deleterious reactions. These side effects can arise from a number of different causes. Polar compounds injected into the CSF are restricted to the CSF, and the distribution of a drug administered i.t. is uneven and incomplete in the CNS. Also, since the rate of distribution is related to the rate of CSF movement, it is often slow. 17 Furthermore, since the ventricular ~5 T. H. Maren, in "Medical Physiology" (V. B. Mountcastle, ed.), p. 1218. Mosby, St. Louis, Missouri, 1980. 16 R. Welch and V. Friedman, Brain 83, 454 (1960). ~7 L. S. Schanker, Antimicrob. Agents Chemother. p. 1044 (1905). is L. D. Prockop, L. S. Schanker, and B. B. Brodie, Science 134, 1424 (1961). 19 H. Davson, J. Physiol. (London) 255, 1 (1976).
1291
TARGETING DRUGS TO BRAIN
385
volume of the CSF is small, increases in intracerebral pressure can occur with repeated injections. Improper needle or catheter placement can result in seizure, encephalitis, tuberculosis, meningitis, arachnoiditis, necrotizing encephalopathy, or cellulitis. The administered compounds themselves can produce toxic reactions. Although macromolecular carriers 2°-22 proved to be of limited use, a general method which can possibly be applied to the delivery of otherwise unusable drugs to the brain is the prodrug approach. 23-27 A major advance in the area of drug delivery to the brain came about as a result of work with the polar quaternary salt, N-methylpyridinium-2carbaldoxime chloride (2-PAM), which is the drug choice in the treatment of organophosphate poisonings. However, 2-PAM as a quarternary pyridinium salt will not pass the BBB and is, therefore, ineffective in treating central intoxication. Bodor et al. 28 approached this problem by transiently removing the positive charge of 2-PAM (1) by chemically reducing the quarternary compound to its corresponding dihydropyridine derivative. This reduction produced a tertiary amine which is far more lipophilic than 2-PAM (Pro-2-PAM; 2). N-Substituted dihydropyridines are known to be relatively unstable and are rapidly oxidized to the parent quarternary compound. This redox system is the basis of the important coenzyme system, NADH ~ NAD.
~ + C----NOH I H CH3 1
~ C I
CH~
: N O H - - H* H
2
~N~ /, I
CH 3
C ]'[OH H
2a
As a result of this, it was found that in vivo pro-2-PAM was dramatically more effective in reactivating phosphorylated enzymes than 2-PAM (Table I). 2oG. Gregoriadis, Nature (London) 265, 407 (1977). 2, G. Gregoriadis, ed., "Drug Carriers in Biology and Medicine." Academic Press, New York, 1979. 22 R. L. Juliano, "Drug Delivery Systems." Oxford Univ. Press, London and New York, 1980. 23 A. A. Sinkula and S. H. Yalkowski, J. Pharm. Sci. 64, 181 (1975). z4 N. Bodor, in "Design of Biopharmaceutical Properties through Prodrugs and Analogs," (E. B. Roche, ed.), p. 98. Am. Pharm. Assoc., Washington, D.C., 1977. 25 N. Bodor, Drugs Future 6, 165 (1981). 26N. Bodor, in "Strategy in Drug Research" (J. A. Buisman, ed.), p. 137. Elsevier, Amsterdam, 1981. 27 V. Stella, in "Prodrugs as Novel Drugs Delivery Systems" (T. Higuchi and V. Stella, eds.), p. 1. Am. Chem. Soc., Washington, D.C., 1975. 28 N. Bodor, E. Shek, and T. Higuchi, Science 190, 155 (1975).
386
PRODRUGS
[29]
TABLE 1 ACTIVITY AND REACTIVATION OF ACETYLCHOLINESTERASE"
Drug
Dose (mg/kg)
Activity (× 106)
SE
Activity (%)
Reactivation (%)
2-PAM 2-PAM 2-PAM Pro-2-PAM Pro-2-PAM Pro-2-PAM Pro-2-PAM
30 40 50 20 30 40 50
1.053 1.683 2.246 2.781 3.111 4.871 7.429
0.076 0.041 0.187 0.163 0.063 0.355 0.183
10.27 16.42 21.92 27.14 30.36 47.53 72.49
0.00 5.93 12.12 18.00 21.63 40.95 69.04
" In m o u s e brain pretreated with D F P and treated minutes later with either 2-PAM (1) or Pro-2-PAM (2a) i.v. Taken from ref. 29.
The loss of the delivered compound from the brain is not as well characterized as its influx. As was discussed earlier, the brain possesses a number of specialized systems for eliminating compounds from the CNS. These include specific molecular pumps which actively remove compounds at the choroid plexus and the simple bulk flow rate of CSF. The efflux, therefore, of a compound from the brain is not precisely proportional to lipophilicity, and 2-PAM is an example of this. The quaternary salt 1 formed in situ is rapidly lost from all locations including the brain. 3° The rapid loss of this quaternary salt from the CNS was attributed to an active process. It was assumed, however, that not all quaternary salts formed in the CNS are good substrates for the active transport system. An appropriately chosen pharmacologically active agent (i.e., one which contains a pyridinium moiety) could be reduced to its corresponding dihydropyridine derivative. 3~ After systemic administration, this lipophilic species will penetrate the BBB as well as into the peripheral tissues. After oxidation to the quaternary salt in all body compartments, it can rapidly be eliminated by renal or biliary mechanisms (kout~). In the CNS, however, the salt, because of its charge, would be retained (ko,~ ~ koL,t2). This yields significant concentration of the compound in the brain relative to the periphery and should increase the efficacy of the compound in the brain while reducing its systemic toxicity (Scheme l). The developed scheme was successfully applied to berberine (3). When dihydroberberine (4) or its hydrochloride were administered i.v., the concentration of berberine found in the brain was large. 32 The ber29 E. 3o N. 3~ N. 32 N.
Shek, T. Higuchi, and N. Bodor, J. Med. Chem., 19, 108, 113 (1976). Bodor, R. G. Roller, and S. S. Selk, J. Pharm. Sci. 67, 685 (1978). Bodor, H. Farag, and M. E. Brewster, Science 214, 1370 (1981). Bodor and M. E. Brewster, Eur. J, Med. Chem. 18, 235 (1983).
[29]
TARGETING DRUGS TO BRAIN
387
BRAIN ~
reduction )
~ ' k outl , BBB 4/
PERIPHERY
kout2
kox SCHEME 1
berine delivered to the brain in this way was slowly eliminated (t,/2 = 11 hr). This is somewhat slower than the bulk flow of CSF, and this difference is attributable to the more complete distribution of berberine in the CNS afforded by its prodrug. The systemically formed (3) was eliminated via the kidney.
red OX
CH:~O
O CH30 ~
OCH:~
"~
~-/
OCH~ 3
The brain specificity of the dihydroberberine ~ berberine system could further be enhanced by a slow infusion because of the differences in the bidirectional flux rates and the oxidation rates in the brain and blood 33 (Table IlL For example, the concentration of berberine in the brain is higher than in any other tissue analyzed at 45 min. On the basis of these results--namely, that large quaternary salts formed in s i t u in the brain are lost slowly while smaller quaternary salts are lost rapidly--a carrier-mediated drug delivery scheme was proposed. According to this, a pharmacologically active agent whose ability to pass the BBB is low is chemically coupled to a pyridinium carrier (for example, an N-substituted nicotinic acid or nicotinamide). After coupling, the drug-carrier complex would be reduced, thus yielding the dihydropyridine. This reduced complex would then be systemically administered. It ~ M. E. Brewster and N. Bodor, J. Parenter. Sci. Technol. 37, 159 (1983).
388
PRODRUGS
[29]
T A B L E 11 BERBERINE CONCENTRATIONS AFTER SLOW INFUSION OF DIHYDROBERBERINE
Concentration Organ
Concentration
(/xg/g) after
(/zg/g)
infusion
iv. bolus
A (/~g/g)
91.8 _+ 20 351.8 _+ 54 210.2 x 14 67.8_+ 11
+44 166 -139 +33
88 315 165 52
+84 -194 - 11)2 +27
30 s i n
Brain Kidney Lung Liver 45 s i n Brain Kidney Lung l,iver
135.95 185.5 71.4 101.2 162.2 121.4 62.8 79.4
-+ -+ -+ +
13 26 10 23
-+ 8 _+ 19 + 6 + 10
would cross the BBB because of its enhanced membrane permeability and also would be distributed elsewhere in the body. In all locations, oxidation (ko0 would occur. The resultant positively charged drug-carrier complex is rapidly eliminated from the periphery by renal and/or biliary processes (ko~,2) while, in the brain, the compound is retained because of its size and charge. The cleavage of the drug (kcleavage) from the oxidized carrier will also occur ubiquitously. If the rate of this cleavage is more rapid than the rate of efflux (i.e., koutJ ~ kcleavage) of the complex from the brain, a sustained release of the drug could be obtained. This concept is shown in Scheme 2. However, when kout2 ~ kcleavage, the periphery will be void of the active D, thereby resulting in significant reduction in toxicity. The dihydropyridine drug complex is not, therefore, a prodrug but rather a pro-pro-...-drug or better stated, a chemical delivery system (CDS). It is becoming increasingly apparent that simple prodrugs cannot, in many cases, solve complex drug delivery problems. This carrier-mediated delivery scheme was successfully applied to phenylethylamine (5), dopamine 34 (6), tryptophan 35 (7),
~ 5
6
and other related neurotransmitters. 34 N. Bodor and H. H. Farag, J. Med. Chem. 26, 528 (1983). 35 N. Bodor and T. Nakamura, unpublished results.
CI~CH--NH COOH
7
2
[29]
TARGETING DRUGS TO BRAIN
389
x-~ R
~redu
R ction
PERIPHERY
BRAIN
x_~ R
X ~ e a vage
~ou~1
M/kcleavage
BBB ~Lkout3
"]~k°ut6
kout4
out2
out5
SCHEME 2
For example, Fig. l shows that when the dihydropyridine derivative (8) of (5) is administered systemically, the concentration of the quaternary derivative (9) slowly rises in the brain, reaching a maximum at 80 min, after which the concentration slowly declines.
CH3
CH3 8
9
The demonstration of "locking in" of a compound to the brain is unique to this scheme and this technique.
390
PRODRUGS
[29]
0.12
0.10
v z
o
0.08
z 0.06
~O ° •
W c_~ Z 0
•
•
o 0.04 r'r £3
OlD
0.02
0 0
210
0
0 I
8/
0
I
I
I00
I
I
140
I
I
180
I
I
220
TIME (Minutes)
FIG. I, The concentration of I-methyl-3-(N-/3-phenethyl)carbamoylpyridiniumsalt in the brain (O) and the blood (O) of rats after systemic administration of 125 mg/kg of the corresponding dihydro derivative. The chemical delivery system was also applied to the very important class of compounds, the steroids. There are a number of clinical situations in which the delivery of sex hormones to the brain is desirable. These include dysfunctions such as impotency and centrally controlled contraception. For example, when the dihydrocarrier-testosterone complex is administered systemically, 36 the quaternary compound is found (as shown in Fig. 2) in the brain. It is "locked in, ''6 thereby providing a sustained release form for the testosterone. Conversely, the quaternary salt, after systemic injection, was rapidly lost from the peripheral circulation (halflife of efflux of 54 rain). Synthetic Examples
N-Nicotinoyldopamine (10). To a pyridine solution containing 11.7 g (0.05 tool) of dopamine hydrobromide and 6.15 g (0.05 tool) of nicotinic acid at 0 ° was added 10.3 g (0.05 mol) of dicyclohexylcarbodiimide (DCC). The reaction mixture was stirred at room temperature for 24 hr, and the 36 N. Bodor and H. H. Farag, J. Pharm. Sci. 73, 385 (1984).
{29]
TARGETINGDRUGS
TOBRA~N
391
70
O O
m
i0.75
6O
1050 ~
50
~O.Z5 ~
13d 0 b.J
C21
~ 40
Lad --I U.I a::
Z
~m I.L o
"~
0.15
30 '~
~ 2o
olo
::k
0.05
IO .-c] EO
40
60
BO
I 100
i 120
J 140
I 160
I
180
T I M E IN M I N U T E S
FIG. 2. Concentration o f testosterone 17-nicotinate-N-methyl cation in the blood (~) and brain (O) of rats and the concentration of released testosterone (Ir) following an administration of 28.2 mg/kg of the testosterone-CDS. Also given is the concentration of testosterone in the brain (ml) and in the blood (0) following the administration of testosterone itself. The concentration of the released testosterone (VI is indicated by the right abscissa; all other concentrations are indicated by the left.
formed dicyclohexylurea was removed by filtration. The pyridine was removed in vacuo, and the residue was crystallized from water at 0 °. The product was isolated by filtration and dried over phosphorous pentoxide. Recrystallization from 2-propanol gave 0.9 g (0.035 mol, 70%) of N-nicotinoyldopamine, m.p. 159-162 °. 3-(N-{[3-[3,4-Bisfpivalyloxy)phenyl]ethyl}carbamoyl)pyridine (11). To a suspension of 5.16 g (0.02 tool) of finely powdered nicotinoyldopamine in 100 ml of chloroform was added 7.23 g (0.06 mob of trimethylacetyl chloride while stirring. The mixture was refluxed for 6 hr and then filtered. The filtrate was washed with water free of chloride ions, washed once with a 5% solution of NaHCO3, and then with water. The chloroform was evaporated, and the residue was chromatographed by using a silica gel G column and 2% methanol in chloroform as the eluant. The first fraction was collected and evaporated, and the residue was crystallized from
W
z
0 rr lad I---
,--0-°
03 LU l'--
::L
392
PROORUGS
[29]
ether-petroleum ether: yield 6.2 g (73%) of a white crystalline solid; m.p. 112-114 °.
1 -Methyl-3-(N-{[3-[3,4-bis(pivalyloxy)phenyl]carbamoyl})pyridinium Iodide (12). To a solution of 5.0 g ( 11.7 mmol) of compound (11) in 20 ml of acetone was added 3.3 g (23.4 mmol) of methyl iodide; the mixture was refluxcd whilc stirring for 6 hr and then cooled. An orange crystalline solid separated; this solid was filtered, washed with cther, and crystallized from acetone-ether: yield 5.6 g (85%).
17fl-[(1,4-Dihydro-l-methyl-3-pyridinylcarbonyl)oxy]androst-4-en-3one (14). To an ice-cold solution of I. I g (2 mmol) of testosterone nicotinate-N-methyi iodide (13) in 150 ml of deaerated 10% aqueous methanol was added 0.67 g (8 mmol) of sodium bicarbonate and 1.37 g (8 mmol) of sodium dithionite. The mixture was stirred for 20 min at room temperature, and the separated pale-yellow material was removed by filtration, washed with water, and dried over P2Os under vacuum to yield 0.82 g (98%) of (14), m.p. 172-175 °. Analytical Methods
Dopamine Delivery. A high-pressure liquid chromatography (HPLC) method was developed for the studies of the degradation of a dihydropyridine derivative. The chromatographic analysis was performed on a component system consisting of Waters Associates Model 6000A solvent delivery system, Model U6K injection, and Model 440 dual-channel absorbance detector operated at 254 and 280 nm. A 30 cm × 3.9-ram (internal diameter) reverse-phase Bondapak C18 column (Waters Associates), operated at ambient temperature, was used for all separations. The mobile phase used for the separation of the dihydropyridine derivative, its degradation products, and oxidation products consisted of a 0.005 M solution of 1-heptanesulfonic acid sodium salt (PIC B-7 Eastman Kodak) in CH3CN-0.01 M aqueous dibasic ammonium phosphate (2.5 : !), at a flow rate of 2.0 ml/min. Testosterone Delivery. An HPLC method was developed for the studies of the degradation of the quaternary (13) and dihydropyridine derivatives (14) of testosterone using the system described earlier. The absorbance detector was operated at 254 nm. A 15 cm × 4.6-mm i.d., 5-/zm particle size, Ultrasphere reverse-phase C18 column, operated at ambient temperature was used for all separations. The mobile phase used for the separation of the dihydropyridine derivative products and the oxidation products consisted of a 0.002 M solution of l-heptanesulfonic acid sodium salt in CH3CN-0.01 M aqueous Na~HPO4 (7 : 3). At a flow rate of 2.0 ml/ min, (13) has a retention time of 12 min and (14) has a retention time of 5
[29]
TARGETING DRUGS TO BRAIN
393
rain. For the analysis of testosterone in the in vivo brain delivery studies, the solvent consisted of a 0.002 M solution of 1-heptanesulfonic acid sodium salt in CHzCN-0.01 M aqueous Na2HPO4 (1 : 1). At a flow rate of 2.0 ml/min, testosterone has a retention time of 3.3 rain and (13) has a retention time of 36.5 rain (very broad peak).
Determination of the Enzymatic Hydrolytic Cleavage and Rate of Oxidation of the Delivery System (15)for Dopamine In Human Plasma. The freshly collected plasma used was obtained at the Civitan Regional Blood Center, Inc. (Gainesville, FL) and contained about 80% plasma diluted with anticoagulant citrate-phosphate-dextrose solution U.S.P. The plasma was stored in a refrigerator and used the next day. One hundred microliters of freshly prepared 0.61 M solution of (15) in methanol was added to 20 ml of plasma, previously equilibrated to 37° in a water bath, and mixed thoroughly to result in an initial concentration of 3.05 x 10 3 tool/liter. One-milliliter samples of plasma were withdrawn from the test medium, added immediately to 5 ml of ice-cold acetonitrile, shaken vigorously, and placed in a freezer. When all samples had been collected, they were centrifuged, and the supernatant fluids were filtered through Whatman No. 1 filter paper and analyzed by HPLC. In Rat Brain Homogenate. The brain homogenate was prepared by the following method. Five Sprague-Dawley rats were killed by decapitation. The brains were removed, weighed (total weight 98.5 g), and homogenized in 49.3 ml of aqueous 0.11 M phosphate buffer, pH 7.4. The homogenate was centrifuged, and supernatant fluid was used for the test. One hundred microliters of a 0.18 M solution of (15) was mixed with 10 ml of homogenate, previously equilibrated to 37° in a water bath, to result in an initial concentration of 1.8 x 10 3 mol/liter. Samples of 1.0 ml we,re withdrawn every 10 rain from the test medium, added immediately to 5 ml of ice-cold acetonitrile, and placed in a freezer. When samples had been collected, they were centrifuged. Each supernatant was filtered through two Whatman No. 1 filter papers and analyzed by HPLC. In Rat Liver Homogenate. The liver homogenate was prepared by the following method. Three Sprague-Dawley rats were killed by decapitation. The livers were removed, weighed, and homogenized in a tissue homogenizer in 0.11 M aqueous phosphate buffer, pH 7.4, to make 20% liver homogenate. The homogenate was centrifuged, and the supernatant was used for the test. One hundred microliters of a 0.1 M solution of (lfi) in methanol was mixed with 20 ml of the homogenate, previously equilibrated to 37° in a water bath, to result in an initial concentration of 9 x 10 4 mol/liter. Samples of 1.0 ml were withdrawn every 5 rain from the
394
PRODRUGS
[29]
test medium, added immediately to 5 ml of ice-cold acetonitrile, shaken vigorously, and placed in a freezer. When all samples had been collected, they were centrifuged, and each supernatant was filtered through Whatman No. I filter paper and analyzed by HPLC.
Determination of the Concentration of the Locked-in Delivery Form of Dopamine in Brain and Blood of Rats after Parental Administration o f (15) Male Sprague-Dawley rats (average weight, 150 g) were used. The rats were anesthetized with an intramuscular injection of Inovar, and the jugular was exposed. Compound (15) was injected intrajugularly in the form of solution in dimethyl sulfoxide at a dose of 50 mg/kg and at a rate of 24 /zl/min by using a calibrated infusion pump. After appropriate time periods, 1 ml of blood was withdrawn from the heart and dropped immediately into a tared tube containing 3 ml of acetonitrile, which was afterward weighed to determine the weight of the blood taken. The animals were perfused with 20 ml of saline solution and decapitated, and the brain was removed. The weighed brain was homogenized with 0.5 ml of distilled water; 3 ml of acetonitrile was added; the mixture was rehomogenized thoroughly, centrifuged, and filtered; and the filtrate was analyzed for the compounds by using the HPLC method. The tubes containing the blood were shaken vigorously, centrifuged, decanted, and also analyzed using the HPLC method. Quantitation was done by using a recovery standard curve obtained by introducing a known amount of the compound in either brain homogenate or blood and then treated in the same manner. Testosterone Studies
Table III shows the results of kinetic studies in biological fluids.
Determination on in Vitro Rates of Oxidation of (14) In Biological Media. One hundred microliters of a freshly prepared 0.024 M solution of (14) in dimethyl sulfoxide was added to 10 ml of plasma, previously equilibrated to 37° in a water bath, and mixed thoroughly to result in an initial concentration of 2.4 × l0 4 tool/liter. One-milliliter samples of plasma were withdrawn every 20 min from the test medium, added immediately to 5 ml of ice-cold acetonitrile, shaken vigorously, and placed in a freezer. When all samples had been collected, they were centrifuged and the supernatants were filtered through nitrocellulose membrane filters (0.45-~m pore size) and analyzed by HPLC, following the appearance of (13).
[29]
TARGETING DRUGS TO BRAIN
395
TABLE 111 KINETICS OF in Vitro OXIDATION OF THE DIHYDROPYRIDINE ESTER (14) I"O THE QUATERNARY DERIVATIVE (15) IN BIOLOGICAL FLUIDS
Medium
k (s i)
80% Plasma 20% Brain homogenate WhoLe blood
8.12 x 10 ~ 1.72 x 10 4 1.74 x 10 4
ll/2 (rain) 142 67 66
r
0.959 0.997 0.997
In R a t Brain Homogenate. Five female Sprague-Dawley rats were decapitated, and the brains were removed, pooled, weighed (total weight 9.2 g), and homogenized in 36.8 ml of aqueous 0.11 M phosphate buffer, pH 7.4. One hundred microliters of 0.024 M solution of (14) in dimethyl sulfoxide was mixed with 20 ml of the homogenate, previously equilibrated to 37 ° in a water bath, to result in an initial concentration of 2.4 x l0 4 tool/liter. Samples of 1.0 ml were withdrawn every 10 rain from the test medium, added immediately to 5 ml of ice-cold acetonitrile, shaken vigorously, and placed in a freezer. When all samples had been collected, they were centrifuged and the supernatants were filtered through nitrocellulose membrane filters (0.45-/xm pore size) and analyzed by HPLC. In Vitro Determination o f the Site-Specific Conversion of(13) to Testosterone A fresh brain homogenate was prepared as previously described. One hundred microliters of a 0.017 M solution of the quaternary compound (13) in methanol was mixed with 10 ml of the brain homogenate, previously equilibrated to 37 °, to result in an initial concentration of 1.7 x 10 -4 M. Samples of 1.0 ml were withdrawn every 20 min from the test medium, added immediately to 5 ml of ice-cold acetonitrile, and placed in a freezer. When all samples had been collected, they were centrifuged, and the supernatant was filtered through a nitrocellulose membrane filter (0.45/zm pore size) and analyzed for the quaternary compound (13).
Conclusions The inability of many potentially useful agents to cross the BBB has limited the treatment of cerebral diseases. The basis of this impermeability is the peculiar way in which cerebral capillaries are fused. This tight joining prevents all but the most lipophilic compounds from passing into
396
PRODRUGS
[29]
the brain parenchyma. Attempts to circumvent this barrier have included direct injection of therapeutic agents into the CSF, but the technique is replete with dangerous side effects. Another method by which a drug can gain access into the brain is by temporarily making the compound more lipophilic. Unfortunately, when this is done indiscriminately, all tissues are exposed to higher levels of the drug. The approach which seems to be the most useful, and demonstrates the most potential, is the dihydropyridine ~- pyridinium redox system. There are two major areas in this technique. The first involves dealing with molecules which contain a pyridinium partial structure (2-PAM, berberine), whereas the second involves using a pyridine carrier to which a drug can be attached (phenethylamine, dopamine, testosterone). Both approaches are based on the ability to transiently convert highly polar molecules (pyridinium salts) to nonpolar species (dihydropyridines). By performing this conversion, the ability of a molecular to pass biologically important membranes can be altered. This method is superior to simple ester- or amide-type prodrugs in that it delivers compounds specifically to the brain, while maintaining a lower peripheral concentration. This increases the efficacy of the agent centrally, while reducing any associated peripheral toxicity. Acknowledgments Financial support fromthe National Institutesof Health (GrantGM 27167), fromOtsuka Pharmaceutical Co., and from Pharmtec, Inc. is gratefullyacknowledged.
TARGETING DRUGS TO BRAIN
381
These results indicate that prodrugs targeted for intestinal peptidases as well as those targeted for intestinal alkaline phosphatase show potential for improving oral drug delivery. For the stable phosphate derivatives, axial enzyme distribution in different species should determine the relative success for these prodrugs. For those prodrugs targeted for intestinal peptidases, prodrug instability and enzyme specificity with respect to targeting to gain transport advantages for oral administration are key considerations for the utility of this approach. Acknowledgments The authors acknowledge the support of NIH Grant No. 1ROIGM3169 for much of the work presented in the Results section and thank Iris Templin for her expert assistance in preparation of the chapter.
[29] T a r g e t i n g o f D r u g s to t h e B r a i n By NICHOLAS B o o o a Introduction The overall membrane transport properties are very important for a drug, as these properties govern the absorption, distribution, and elimination of its intact form, and affect binding, affinity, and other important characteristics. It is of even greater importance to find methods which enable delivery of drugs specifically to a particular organ, or site. This requires more than simply optimizing overall membrane transport characteristics. Among the various possible ways to achieve site-specific or organ-specific delivery, the chemical delivery system (CDS) is the most flexible)--and it offers possibilities for specific delivery not only to the brain, or eye 2 but to other organs and sites. Properly designed, a CDS or any other drug delivery system should concentrate the desired active agent at its site of action and reduce its concentration in other locations. The main result of this manipulation is not only an increase in the efficacy of the drug entity but also a decrease in its toxicity. A site-specific delivery designed for the central nervous system (CNS) would be especially useful. It is well known that many of the pharmacologically active and t N. Bodor and H. H. Farag, J. Med. Chem. 25, 313 (1983). 2 N. Bodor and G. Visor, Exp. Eye Res. 38, 621 (1984).
METHODS IN ENZYMOLOGY,VOL. 112
Copyright © 1985by Academic Press, Inc. All rightsof reproductionin any form reserved. 1SBN 0-12-182012-2
382
PRODRUGS
[29]
important agents, including endogenous neurotransmitters, cannot be transported from the blood into the brain because of a set of specialized barriers present at the blood-brain interface. This barrier system is generally called the blood-brain barrier (BBB) and is composed of a set of anatomical and enzymatic components. The existence of a barrier which separates the general circulation from the central nervous system was first postulated by Ehrlich at the end of the nineteenth century. 3-5 The actual barrier was later described as acting against complexes formed between the dyes and plasma proteins with which these compounds are extensively bound. However, it was discovered that many small compounds are also unable to pass this barrier. The morphological basis of the BBB was for long a rather controversial issue. Most recently, the anatomical basis for the BBB was identified as the endothelial lining of the cerebral capillaries, and most importantly, not the perivascular cells. There are several ultrastructural differences between systemic capillaries and cerebral capillaries which explain the difference in their permeabilities. The main difference is in the manner in which endothelial cells in cerebral capillaries are joined. Cerebral junctions are characterized as tight or closed junctions which gird the cell circumferentially, forming zona occludens and providing an absolute barrier. Structurally, the junctions consist of aligned intramembranous ridges and grooves which are in close apposition. 6 Systemic capillaries lack this closed junction. Morphologically, this can be traced to a lack of continuity in the intercellular appositions. Materials pass easily between these leaky cells, while in the brain the sealing of the intercellular fissures severely restricts this nonspecific transport. Since intercellular transport is impossible, only intracellular or transcellular transport remains. Lipophilic compounds can readily pass through these phospholipoidal membranes, but hydrophilic compounds and high-molecular-weight substances are excluded. A second main difference between cerebral and systemic capillaries is the paucity of vesicles and vesicular transport in the CNS.7 A third difference is the lack of fenestrae in the cerebral capillaries. Cerebral vessels have a number of perivascular accessory structures which appear to be involved in the function of the BBB. Astrocytic foot processes may be involved in the regulation of the amino acid flux. Phago3 W. M. Pardridge, J. D. Conner, and I. L. Crawford, CRC Crit. Rev. Toxicol. 3, 159 (1975). 4 E. Levin, Exp. Eye. Res. 25, Suppl., 191 (1977). 5 B. Van Deurs, Int. Rev. Cytol. 65, 117 (1980). M. W. Brightman and T. S. Reese, J. Cell Biol. 40, 648 (1969). 7 M. W. Brightman, Exp. Eye Res. 25, Suppl., 1 (1977).
[29]
TARGETING DRUGS TO BRAIN
383
cytic pericytes which are present abluminally may play a similar role. It is possible that the basement membrane of endothelial capillaries acts as a mass filter, preventing large molecules from penetrating it. In addition to these structural features, the BBB maintains a number of enzymes which appear to augment barrier function. 4,8.9 Optimal neuronal control requires a careful balancing between neurotransmitter release, metabolism, and uptake, as it is of vital importance to restrict the entry of blood-borne neurotransmitters into the CNS. This is why high concentrations of such enzymes as catechol O-methyltransfcrase (COMT), monoamine oxidase (MAO), 3;-aminobutyric acid aminotransferase (GABA-T), and aromatic-L-amino-acid decarboxylase (DOPA-decarboxylase) are found in the BBB. The enzymatic BBB may also play a role in the exclusion of some lipophilic compounds which otherwise might passively diffuse through the barrier. The transport of nutrients is brought about by a number of carriers which are situated in the endothelial cells and which are assumed to be proteinaceous materials. They are equilibrative, i.e., nonenergy dependent and bidirectional in nature, and can be saturated, m.jl The net movement of compounds is always along a concentration gradient, and since nutrients are readily utilized as soon as they pass into the brain, this gradient is in the direction of the brain. The BBB, therefore, consists of a relatively impermeable membrane superimposed on which are mechanisms for allowing the entrance of essential nutrients and the exit of metabolic wastes. If a compound is to gain access to brain parenchyma, it may do so via several routes. If the molecule has affinity for one of the carriers previously described, it may diffuse across the BBB by association with this carrier. A compound with high intrinsic lipophilicity can diffuse passively through the phospholipid cell membrane matrix. These two avenues, namely, passive diffusion and carrier mediation, represent the major components for influx. Other minor mechanisms may also allow the entry of substrates to the C N S . 7']2 Retrograde axoplasmic transport has been observed in such areas as the nucleus ambiguous and the abducens nucleus. There are several areas in the brain which lack a BBB. 13,~4 These J. E. Hardebo and B. Nilsson, Acta Physiol. Stand. 107, 153 (1979). 9 j. E. Bardebo and C. O w m a n , Ann. Neurol. 8, 1 (1980). l0 W. M. Pardridge, Diabetologia 20, 246 (1981). " W. M. Pardridge and W. H. Oldendor, J. Neurochem. 28, 5 (1977). ~z E. Westergaard, Adv. Neurol. 28, 55 (1980). 13 C. W. Wilson and B. B. Brodie, J. Pharmacol. Exp. Ther. 133, 332 (1961). t4 G. D. Pappas, J. Neurol. Sci. 10, 241 (1970).
384
PRODRUGS
[291
include locations near the ventricles such as the area postrema, the subfornical organ, the median eminence of the neurohypophysis, the organum vasculosum of the lamina terminalis, and the choroid plexus. Collectively, these areas are termed the circumventricular organ. In addition, the pineal gland also lacks a BBB. These areas constitute a small fraction of the total surface area of the BBB and may allow a limited nonspecific flux. Cerebral spinal fluid (CSF) is produced at the choroid plexus and drains from the ventricles through the foramina of Magendie and Luschka into the subarachnoid space of the brain, j5 Cerebral spinal fluid, along with any dissolved materials, leaves the subaraehnoid space via the arachnoid villi, which protrude into a venous sinus. The arachnoid villi act as a one-way valve and prevent backflow. 16 This loss of CSF provides a slow mechanism for nonspecific efflux of compounds from the CNS. The mechanism rids the brain of polar compounds such as metabolic wastes at a fairly constant rate regardless of the molecular size. Therefore, while lipophilicity is very important for influx to the brain, the efflux of a compound is only partially dependent on this parameter. 17-19 The BBB excludes a number of pharmacologically active agents and, as such, treatment of many cerebral diseases is severely limited. To increase the effectiveness of drugs which are active against these diseases, the specific transit time of a desired drug in the brain must be increased. This should increase the therapeutic index of an agent since not only is the concentration and/or the residence time of the agent increased in the vicinity of the bioreceptor, but of equal importance, the peripheral concentration of the drug is reduced, thereby decreasing any associated toxicity. Unfortunately, there are very few methods for circumventing the BBB, and these are of limited usefulness. The direct administration of drugs in the CNS, i.e., an intrathecal injection (i.t.), has been used to deliver various pharmacologically active agents. While i.t. injections are of potential value, they are notorious for their high incidence of deleterious reactions. These side effects can arise from a number of different causes. Polar compounds injected into the CSF are restricted to the CSF, and the distribution of a drug administered i.t. is uneven and incomplete in the CNS. Also, since the rate of distribution is related to the rate of CSF movement, it is often slow. 17 Furthermore, since the ventricular ~5 T. H. Maren, in "Medical Physiology" (V. B. Mountcastle, ed.), p. 1218. Mosby, St. Louis, Missouri, 1980. 16 R. Welch and V. Friedman, Brain 83, 454 (1960). ~7 L. S. Schanker, Antimicrob. Agents Chemother. p. 1044 (1905). is L. D. Prockop, L. S. Schanker, and B. B. Brodie, Science 134, 1424 (1961). 19 H. Davson, J. Physiol. (London) 255, 1 (1976).
1291
TARGETING DRUGS TO BRAIN
385
volume of the CSF is small, increases in intracerebral pressure can occur with repeated injections. Improper needle or catheter placement can result in seizure, encephalitis, tuberculosis, meningitis, arachnoiditis, necrotizing encephalopathy, or cellulitis. The administered compounds themselves can produce toxic reactions. Although macromolecular carriers 2°-22 proved to be of limited use, a general method which can possibly be applied to the delivery of otherwise unusable drugs to the brain is the prodrug approach. 23-27 A major advance in the area of drug delivery to the brain came about as a result of work with the polar quaternary salt, N-methylpyridinium-2carbaldoxime chloride (2-PAM), which is the drug choice in the treatment of organophosphate poisonings. However, 2-PAM as a quarternary pyridinium salt will not pass the BBB and is, therefore, ineffective in treating central intoxication. Bodor et al. 28 approached this problem by transiently removing the positive charge of 2-PAM (1) by chemically reducing the quarternary compound to its corresponding dihydropyridine derivative. This reduction produced a tertiary amine which is far more lipophilic than 2-PAM (Pro-2-PAM; 2). N-Substituted dihydropyridines are known to be relatively unstable and are rapidly oxidized to the parent quarternary compound. This redox system is the basis of the important coenzyme system, NADH ~ NAD.
~ + C----NOH I H CH3 1
~ C I
CH~
: N O H - - H* H
2
~N~ /, I
CH 3
C ]'[OH H
2a
As a result of this, it was found that in vivo pro-2-PAM was dramatically more effective in reactivating phosphorylated enzymes than 2-PAM (Table I). 2oG. Gregoriadis, Nature (London) 265, 407 (1977). 2, G. Gregoriadis, ed., "Drug Carriers in Biology and Medicine." Academic Press, New York, 1979. 22 R. L. Juliano, "Drug Delivery Systems." Oxford Univ. Press, London and New York, 1980. 23 A. A. Sinkula and S. H. Yalkowski, J. Pharm. Sci. 64, 181 (1975). z4 N. Bodor, in "Design of Biopharmaceutical Properties through Prodrugs and Analogs," (E. B. Roche, ed.), p. 98. Am. Pharm. Assoc., Washington, D.C., 1977. 25 N. Bodor, Drugs Future 6, 165 (1981). 26N. Bodor, in "Strategy in Drug Research" (J. A. Buisman, ed.), p. 137. Elsevier, Amsterdam, 1981. 27 V. Stella, in "Prodrugs as Novel Drugs Delivery Systems" (T. Higuchi and V. Stella, eds.), p. 1. Am. Chem. Soc., Washington, D.C., 1975. 28 N. Bodor, E. Shek, and T. Higuchi, Science 190, 155 (1975).
386
PRODRUGS
[29]
TABLE 1 ACTIVITY AND REACTIVATION OF ACETYLCHOLINESTERASE"
Drug
Dose (mg/kg)
Activity (× 106)
SE
Activity (%)
Reactivation (%)
2-PAM 2-PAM 2-PAM Pro-2-PAM Pro-2-PAM Pro-2-PAM Pro-2-PAM
30 40 50 20 30 40 50
1.053 1.683 2.246 2.781 3.111 4.871 7.429
0.076 0.041 0.187 0.163 0.063 0.355 0.183
10.27 16.42 21.92 27.14 30.36 47.53 72.49
0.00 5.93 12.12 18.00 21.63 40.95 69.04
" In m o u s e brain pretreated with D F P and treated minutes later with either 2-PAM (1) or Pro-2-PAM (2a) i.v. Taken from ref. 29.
The loss of the delivered compound from the brain is not as well characterized as its influx. As was discussed earlier, the brain possesses a number of specialized systems for eliminating compounds from the CNS. These include specific molecular pumps which actively remove compounds at the choroid plexus and the simple bulk flow rate of CSF. The efflux, therefore, of a compound from the brain is not precisely proportional to lipophilicity, and 2-PAM is an example of this. The quaternary salt 1 formed in situ is rapidly lost from all locations including the brain. 3° The rapid loss of this quaternary salt from the CNS was attributed to an active process. It was assumed, however, that not all quaternary salts formed in the CNS are good substrates for the active transport system. An appropriately chosen pharmacologically active agent (i.e., one which contains a pyridinium moiety) could be reduced to its corresponding dihydropyridine derivative. 3~ After systemic administration, this lipophilic species will penetrate the BBB as well as into the peripheral tissues. After oxidation to the quaternary salt in all body compartments, it can rapidly be eliminated by renal or biliary mechanisms (kout~). In the CNS, however, the salt, because of its charge, would be retained (ko,~ ~ koL,t2). This yields significant concentration of the compound in the brain relative to the periphery and should increase the efficacy of the compound in the brain while reducing its systemic toxicity (Scheme l). The developed scheme was successfully applied to berberine (3). When dihydroberberine (4) or its hydrochloride were administered i.v., the concentration of berberine found in the brain was large. 32 The ber29 E. 3o N. 3~ N. 32 N.
Shek, T. Higuchi, and N. Bodor, J. Med. Chem., 19, 108, 113 (1976). Bodor, R. G. Roller, and S. S. Selk, J. Pharm. Sci. 67, 685 (1978). Bodor, H. Farag, and M. E. Brewster, Science 214, 1370 (1981). Bodor and M. E. Brewster, Eur. J, Med. Chem. 18, 235 (1983).
[29]
TARGETING DRUGS TO BRAIN
387
BRAIN ~
reduction )
~ ' k outl , BBB 4/
PERIPHERY
kout2
kox SCHEME 1
berine delivered to the brain in this way was slowly eliminated (t,/2 = 11 hr). This is somewhat slower than the bulk flow of CSF, and this difference is attributable to the more complete distribution of berberine in the CNS afforded by its prodrug. The systemically formed (3) was eliminated via the kidney.
red OX
CH:~O
O CH30 ~
OCH:~
"~
~-/
OCH~ 3
The brain specificity of the dihydroberberine ~ berberine system could further be enhanced by a slow infusion because of the differences in the bidirectional flux rates and the oxidation rates in the brain and blood 33 (Table IlL For example, the concentration of berberine in the brain is higher than in any other tissue analyzed at 45 min. On the basis of these results--namely, that large quaternary salts formed in s i t u in the brain are lost slowly while smaller quaternary salts are lost rapidly--a carrier-mediated drug delivery scheme was proposed. According to this, a pharmacologically active agent whose ability to pass the BBB is low is chemically coupled to a pyridinium carrier (for example, an N-substituted nicotinic acid or nicotinamide). After coupling, the drug-carrier complex would be reduced, thus yielding the dihydropyridine. This reduced complex would then be systemically administered. It ~ M. E. Brewster and N. Bodor, J. Parenter. Sci. Technol. 37, 159 (1983).
388
PRODRUGS
[29]
T A B L E 11 BERBERINE CONCENTRATIONS AFTER SLOW INFUSION OF DIHYDROBERBERINE
Concentration Organ
Concentration
(/xg/g) after
(/zg/g)
infusion
iv. bolus
A (/~g/g)
91.8 _+ 20 351.8 _+ 54 210.2 x 14 67.8_+ 11
+44 166 -139 +33
88 315 165 52
+84 -194 - 11)2 +27
30 s i n
Brain Kidney Lung Liver 45 s i n Brain Kidney Lung l,iver
135.95 185.5 71.4 101.2 162.2 121.4 62.8 79.4
-+ -+ -+ +
13 26 10 23
-+ 8 _+ 19 + 6 + 10
would cross the BBB because of its enhanced membrane permeability and also would be distributed elsewhere in the body. In all locations, oxidation (ko0 would occur. The resultant positively charged drug-carrier complex is rapidly eliminated from the periphery by renal and/or biliary processes (ko~,2) while, in the brain, the compound is retained because of its size and charge. The cleavage of the drug (kcleavage) from the oxidized carrier will also occur ubiquitously. If the rate of this cleavage is more rapid than the rate of efflux (i.e., koutJ ~ kcleavage) of the complex from the brain, a sustained release of the drug could be obtained. This concept is shown in Scheme 2. However, when kout2 ~ kcleavage, the periphery will be void of the active D, thereby resulting in significant reduction in toxicity. The dihydropyridine drug complex is not, therefore, a prodrug but rather a pro-pro-...-drug or better stated, a chemical delivery system (CDS). It is becoming increasingly apparent that simple prodrugs cannot, in many cases, solve complex drug delivery problems. This carrier-mediated delivery scheme was successfully applied to phenylethylamine (5), dopamine 34 (6), tryptophan 35 (7),
~ 5
6
and other related neurotransmitters. 34 N. Bodor and H. H. Farag, J. Med. Chem. 26, 528 (1983). 35 N. Bodor and T. Nakamura, unpublished results.
CI~CH--NH COOH
7
2
[29]
TARGETING DRUGS TO BRAIN
389
x-~ R
~redu
R ction
PERIPHERY
BRAIN
x_~ R
X ~ e a vage
~ou~1
M/kcleavage
BBB ~Lkout3
"]~k°ut6
kout4
out2
out5
SCHEME 2
For example, Fig. l shows that when the dihydropyridine derivative (8) of (5) is administered systemically, the concentration of the quaternary derivative (9) slowly rises in the brain, reaching a maximum at 80 min, after which the concentration slowly declines.
CH3
CH3 8
9
The demonstration of "locking in" of a compound to the brain is unique to this scheme and this technique.
390
PRODRUGS
[29]
0.12
0.10
v z
o
0.08
z 0.06
~O ° •
W c_~ Z 0
•
•
o 0.04 r'r £3
OlD
0.02
0 0
210
0
0 I
8/
0
I
I
I00
I
I
140
I
I
180
I
I
220
TIME (Minutes)
FIG. I, The concentration of I-methyl-3-(N-/3-phenethyl)carbamoylpyridiniumsalt in the brain (O) and the blood (O) of rats after systemic administration of 125 mg/kg of the corresponding dihydro derivative. The chemical delivery system was also applied to the very important class of compounds, the steroids. There are a number of clinical situations in which the delivery of sex hormones to the brain is desirable. These include dysfunctions such as impotency and centrally controlled contraception. For example, when the dihydrocarrier-testosterone complex is administered systemically, 36 the quaternary compound is found (as shown in Fig. 2) in the brain. It is "locked in, ''6 thereby providing a sustained release form for the testosterone. Conversely, the quaternary salt, after systemic injection, was rapidly lost from the peripheral circulation (halflife of efflux of 54 rain). Synthetic Examples
N-Nicotinoyldopamine (10). To a pyridine solution containing 11.7 g (0.05 tool) of dopamine hydrobromide and 6.15 g (0.05 tool) of nicotinic acid at 0 ° was added 10.3 g (0.05 mol) of dicyclohexylcarbodiimide (DCC). The reaction mixture was stirred at room temperature for 24 hr, and the 36 N. Bodor and H. H. Farag, J. Pharm. Sci. 73, 385 (1984).
{29]
TARGETINGDRUGS
TOBRA~N
391
70
O O
m
i0.75
6O
1050 ~
50
~O.Z5 ~
13d 0 b.J
C21
~ 40
Lad --I U.I a::
Z
~m I.L o
"~
0.15
30 '~
~ 2o
olo
::k
0.05
IO .-c] EO
40
60
BO
I 100
i 120
J 140
I 160
I
180
T I M E IN M I N U T E S
FIG. 2. Concentration o f testosterone 17-nicotinate-N-methyl cation in the blood (~) and brain (O) of rats and the concentration of released testosterone (Ir) following an administration of 28.2 mg/kg of the testosterone-CDS. Also given is the concentration of testosterone in the brain (ml) and in the blood (0) following the administration of testosterone itself. The concentration of the released testosterone (VI is indicated by the right abscissa; all other concentrations are indicated by the left.
formed dicyclohexylurea was removed by filtration. The pyridine was removed in vacuo, and the residue was crystallized from water at 0 °. The product was isolated by filtration and dried over phosphorous pentoxide. Recrystallization from 2-propanol gave 0.9 g (0.035 mol, 70%) of N-nicotinoyldopamine, m.p. 159-162 °. 3-(N-{[3-[3,4-Bisfpivalyloxy)phenyl]ethyl}carbamoyl)pyridine (11). To a suspension of 5.16 g (0.02 tool) of finely powdered nicotinoyldopamine in 100 ml of chloroform was added 7.23 g (0.06 mob of trimethylacetyl chloride while stirring. The mixture was refluxed for 6 hr and then filtered. The filtrate was washed with water free of chloride ions, washed once with a 5% solution of NaHCO3, and then with water. The chloroform was evaporated, and the residue was chromatographed by using a silica gel G column and 2% methanol in chloroform as the eluant. The first fraction was collected and evaporated, and the residue was crystallized from
W
z
0 rr lad I---
,--0-°
03 LU l'--
::L
392
PROORUGS
[29]
ether-petroleum ether: yield 6.2 g (73%) of a white crystalline solid; m.p. 112-114 °.
1 -Methyl-3-(N-{[3-[3,4-bis(pivalyloxy)phenyl]carbamoyl})pyridinium Iodide (12). To a solution of 5.0 g ( 11.7 mmol) of compound (11) in 20 ml of acetone was added 3.3 g (23.4 mmol) of methyl iodide; the mixture was refluxcd whilc stirring for 6 hr and then cooled. An orange crystalline solid separated; this solid was filtered, washed with cther, and crystallized from acetone-ether: yield 5.6 g (85%).
17fl-[(1,4-Dihydro-l-methyl-3-pyridinylcarbonyl)oxy]androst-4-en-3one (14). To an ice-cold solution of I. I g (2 mmol) of testosterone nicotinate-N-methyi iodide (13) in 150 ml of deaerated 10% aqueous methanol was added 0.67 g (8 mmol) of sodium bicarbonate and 1.37 g (8 mmol) of sodium dithionite. The mixture was stirred for 20 min at room temperature, and the separated pale-yellow material was removed by filtration, washed with water, and dried over P2Os under vacuum to yield 0.82 g (98%) of (14), m.p. 172-175 °. Analytical Methods
Dopamine Delivery. A high-pressure liquid chromatography (HPLC) method was developed for the studies of the degradation of a dihydropyridine derivative. The chromatographic analysis was performed on a component system consisting of Waters Associates Model 6000A solvent delivery system, Model U6K injection, and Model 440 dual-channel absorbance detector operated at 254 and 280 nm. A 30 cm × 3.9-ram (internal diameter) reverse-phase Bondapak C18 column (Waters Associates), operated at ambient temperature, was used for all separations. The mobile phase used for the separation of the dihydropyridine derivative, its degradation products, and oxidation products consisted of a 0.005 M solution of 1-heptanesulfonic acid sodium salt (PIC B-7 Eastman Kodak) in CH3CN-0.01 M aqueous dibasic ammonium phosphate (2.5 : !), at a flow rate of 2.0 ml/min. Testosterone Delivery. An HPLC method was developed for the studies of the degradation of the quaternary (13) and dihydropyridine derivatives (14) of testosterone using the system described earlier. The absorbance detector was operated at 254 nm. A 15 cm × 4.6-mm i.d., 5-/zm particle size, Ultrasphere reverse-phase C18 column, operated at ambient temperature was used for all separations. The mobile phase used for the separation of the dihydropyridine derivative products and the oxidation products consisted of a 0.002 M solution of l-heptanesulfonic acid sodium salt in CH3CN-0.01 M aqueous Na~HPO4 (7 : 3). At a flow rate of 2.0 ml/ min, (13) has a retention time of 12 min and (14) has a retention time of 5
[29]
TARGETING DRUGS TO BRAIN
393
rain. For the analysis of testosterone in the in vivo brain delivery studies, the solvent consisted of a 0.002 M solution of 1-heptanesulfonic acid sodium salt in CHzCN-0.01 M aqueous Na2HPO4 (1 : 1). At a flow rate of 2.0 ml/min, testosterone has a retention time of 3.3 rain and (13) has a retention time of 36.5 rain (very broad peak).
Determination of the Enzymatic Hydrolytic Cleavage and Rate of Oxidation of the Delivery System (15)for Dopamine In Human Plasma. The freshly collected plasma used was obtained at the Civitan Regional Blood Center, Inc. (Gainesville, FL) and contained about 80% plasma diluted with anticoagulant citrate-phosphate-dextrose solution U.S.P. The plasma was stored in a refrigerator and used the next day. One hundred microliters of freshly prepared 0.61 M solution of (15) in methanol was added to 20 ml of plasma, previously equilibrated to 37° in a water bath, and mixed thoroughly to result in an initial concentration of 3.05 x 10 3 tool/liter. One-milliliter samples of plasma were withdrawn from the test medium, added immediately to 5 ml of ice-cold acetonitrile, shaken vigorously, and placed in a freezer. When all samples had been collected, they were centrifuged, and the supernatant fluids were filtered through Whatman No. 1 filter paper and analyzed by HPLC. In Rat Brain Homogenate. The brain homogenate was prepared by the following method. Five Sprague-Dawley rats were killed by decapitation. The brains were removed, weighed (total weight 98.5 g), and homogenized in 49.3 ml of aqueous 0.11 M phosphate buffer, pH 7.4. The homogenate was centrifuged, and supernatant fluid was used for the test. One hundred microliters of a 0.18 M solution of (15) was mixed with 10 ml of homogenate, previously equilibrated to 37° in a water bath, to result in an initial concentration of 1.8 x 10 3 mol/liter. Samples of 1.0 ml we,re withdrawn every 10 rain from the test medium, added immediately to 5 ml of ice-cold acetonitrile, and placed in a freezer. When samples had been collected, they were centrifuged. Each supernatant was filtered through two Whatman No. 1 filter papers and analyzed by HPLC. In Rat Liver Homogenate. The liver homogenate was prepared by the following method. Three Sprague-Dawley rats were killed by decapitation. The livers were removed, weighed, and homogenized in a tissue homogenizer in 0.11 M aqueous phosphate buffer, pH 7.4, to make 20% liver homogenate. The homogenate was centrifuged, and the supernatant was used for the test. One hundred microliters of a 0.1 M solution of (lfi) in methanol was mixed with 20 ml of the homogenate, previously equilibrated to 37° in a water bath, to result in an initial concentration of 9 x 10 4 mol/liter. Samples of 1.0 ml were withdrawn every 5 rain from the
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test medium, added immediately to 5 ml of ice-cold acetonitrile, shaken vigorously, and placed in a freezer. When all samples had been collected, they were centrifuged, and each supernatant was filtered through Whatman No. I filter paper and analyzed by HPLC.
Determination of the Concentration of the Locked-in Delivery Form of Dopamine in Brain and Blood of Rats after Parental Administration o f (15) Male Sprague-Dawley rats (average weight, 150 g) were used. The rats were anesthetized with an intramuscular injection of Inovar, and the jugular was exposed. Compound (15) was injected intrajugularly in the form of solution in dimethyl sulfoxide at a dose of 50 mg/kg and at a rate of 24 /zl/min by using a calibrated infusion pump. After appropriate time periods, 1 ml of blood was withdrawn from the heart and dropped immediately into a tared tube containing 3 ml of acetonitrile, which was afterward weighed to determine the weight of the blood taken. The animals were perfused with 20 ml of saline solution and decapitated, and the brain was removed. The weighed brain was homogenized with 0.5 ml of distilled water; 3 ml of acetonitrile was added; the mixture was rehomogenized thoroughly, centrifuged, and filtered; and the filtrate was analyzed for the compounds by using the HPLC method. The tubes containing the blood were shaken vigorously, centrifuged, decanted, and also analyzed using the HPLC method. Quantitation was done by using a recovery standard curve obtained by introducing a known amount of the compound in either brain homogenate or blood and then treated in the same manner. Testosterone Studies
Table III shows the results of kinetic studies in biological fluids.
Determination on in Vitro Rates of Oxidation of (14) In Biological Media. One hundred microliters of a freshly prepared 0.024 M solution of (14) in dimethyl sulfoxide was added to 10 ml of plasma, previously equilibrated to 37° in a water bath, and mixed thoroughly to result in an initial concentration of 2.4 × l0 4 tool/liter. One-milliliter samples of plasma were withdrawn every 20 min from the test medium, added immediately to 5 ml of ice-cold acetonitrile, shaken vigorously, and placed in a freezer. When all samples had been collected, they were centrifuged and the supernatants were filtered through nitrocellulose membrane filters (0.45-~m pore size) and analyzed by HPLC, following the appearance of (13).
[29]
TARGETING DRUGS TO BRAIN
395
TABLE 111 KINETICS OF in Vitro OXIDATION OF THE DIHYDROPYRIDINE ESTER (14) I"O THE QUATERNARY DERIVATIVE (15) IN BIOLOGICAL FLUIDS
Medium
k (s i)
80% Plasma 20% Brain homogenate WhoLe blood
8.12 x 10 ~ 1.72 x 10 4 1.74 x 10 4
ll/2 (rain) 142 67 66
r
0.959 0.997 0.997
In R a t Brain Homogenate. Five female Sprague-Dawley rats were decapitated, and the brains were removed, pooled, weighed (total weight 9.2 g), and homogenized in 36.8 ml of aqueous 0.11 M phosphate buffer, pH 7.4. One hundred microliters of 0.024 M solution of (14) in dimethyl sulfoxide was mixed with 20 ml of the homogenate, previously equilibrated to 37 ° in a water bath, to result in an initial concentration of 2.4 x l0 4 tool/liter. Samples of 1.0 ml were withdrawn every 10 rain from the test medium, added immediately to 5 ml of ice-cold acetonitrile, shaken vigorously, and placed in a freezer. When all samples had been collected, they were centrifuged and the supernatants were filtered through nitrocellulose membrane filters (0.45-/xm pore size) and analyzed by HPLC. In Vitro Determination o f the Site-Specific Conversion of(13) to Testosterone A fresh brain homogenate was prepared as previously described. One hundred microliters of a 0.017 M solution of the quaternary compound (13) in methanol was mixed with 10 ml of the brain homogenate, previously equilibrated to 37 °, to result in an initial concentration of 1.7 x 10 -4 M. Samples of 1.0 ml were withdrawn every 20 min from the test medium, added immediately to 5 ml of ice-cold acetonitrile, and placed in a freezer. When all samples had been collected, they were centrifuged, and the supernatant was filtered through a nitrocellulose membrane filter (0.45/zm pore size) and analyzed for the quaternary compound (13).
Conclusions The inability of many potentially useful agents to cross the BBB has limited the treatment of cerebral diseases. The basis of this impermeability is the peculiar way in which cerebral capillaries are fused. This tight joining prevents all but the most lipophilic compounds from passing into
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the brain parenchyma. Attempts to circumvent this barrier have included direct injection of therapeutic agents into the CSF, but the technique is replete with dangerous side effects. Another method by which a drug can gain access into the brain is by temporarily making the compound more lipophilic. Unfortunately, when this is done indiscriminately, all tissues are exposed to higher levels of the drug. The approach which seems to be the most useful, and demonstrates the most potential, is the dihydropyridine ~- pyridinium redox system. There are two major areas in this technique. The first involves dealing with molecules which contain a pyridinium partial structure (2-PAM, berberine), whereas the second involves using a pyridine carrier to which a drug can be attached (phenethylamine, dopamine, testosterone). Both approaches are based on the ability to transiently convert highly polar molecules (pyridinium salts) to nonpolar species (dihydropyridines). By performing this conversion, the ability of a molecular to pass biologically important membranes can be altered. This method is superior to simple ester- or amide-type prodrugs in that it delivers compounds specifically to the brain, while maintaining a lower peripheral concentration. This increases the efficacy of the agent centrally, while reducing any associated peripheral toxicity. Acknowledgments Financial support fromthe National Institutesof Health (GrantGM 27167), fromOtsuka Pharmaceutical Co., and from Pharmtec, Inc. is gratefullyacknowledged.