Controlled release of anti-cocaine catalytic antibody from biodegradable polymer microspheres.

Recent reports have shown that anti-cocaine catalytic monoclonal antibody 15A10 reduces the toxic effect of cocaine by increasing its breakdown to systemically inert products ecgonine methylester and benzoic acid. This study reports the microencapsulation of antibody 15A10 using biodegradable poly (lactic-glycolic) acid (PLGA) by double emulsion technique. Formulation parameters such as protein loading, polymer molecular weight and the presence of zinc carbonate were studied for their effects on in-vitro release of antibody from microspheres. The initial burst release was decreased by the reduction of the protein (as % of total ingredients) in the formulation. Although changing the polymer molecular weight did not cause a reduction in initial burst release, it was effective in improving the release rate. The inclusion of zinc carbonate in microsphere preparation resulted in increase in initial burst release. An in-vivo study in mice revealed the presence of antibody in blood up to ten days following subcutaneous injections. These data demonstrate a potential for a sustained-release formulation of monoclonal antibody 15A10 for treatment of cocaine addiction.

Cortical impact injury in rats promotes a rapid and sustained increase in polyunsaturated free fatty acids and diacylglycerols.

Neurotrauma activates the release of membrane phospholipid-derived second messengers, such as free arachidonic acid (20:4n-6, AA) and diacylglycerols (DAGs). In the present study, we analyze the effect of cortical impact injury of low-grade severity applied to the rat frontal right sensory-motor cortex (FRC) on the accumulation of free fatty acids (FFAs) and DAGs in eight brain areas 30 min and 24 hours after the insult. At these times, accumulation of FFAs and DAGs occurred mainly in the damaged FRC. The cerebellum was the only other brain area that displayed a significant accumulation of DAGs by day one post-injury. By 30 min, accumulation of free AA in the FRC displayed the greatest relative increase (300% over sham value), followed by free docosahexaenoic acid (22:6n-3, DHA, 150%), while both 20:4-DAGs and 22:6-DAGs were increased 100% over sham values. At day one, free 22:6 and 22:6-DAGs showed the greatest increase (590% and 230%, respectively). These results suggest that TBI elicits the hydrolysis of phospholipids enriched in excitable membranes, targeting early on 20:4-phospholipids (by 30 min post- trauma) and followed 24 hours later by preferential hydrolysis of DHA-phospholipids. These lipid metabolic changes may contribute to the initiation and maturation of neuronal and fiber track degeneration observed following cortical impact injury.

Phase I/II trial of all-trans retinoic acid and tamoxifen in patients with advanced breast cancer.

Because tamoxifen and all-trans-retinoic acid (ATRA) have additive antitumor effects in preclinical systems, we performed a Phase I/II clinical trial of this combination in patients with advanced breast cancer. Patients with potentially hormone-responsive advanced breast cancer were enrolled. All received 20 mg of tamoxifen by mouth daily. Consecutive cohorts of 3-6 patients were treated on odd-numbered weeks with ATRA at doses of 70, 110, 150, 190, or 230 mg/m2/day. Twenty-six patients were entered in this trial; 25 were evaluable. A dose of 230 mg/m2 ATRA produced unacceptable headache and dermatological toxicity, but doses < or = 190 mg/m2 were tolerable. Two of 7 patients with measurable disease responded. Seven of 18 patients with evaluable, nonmeasurable disease achieved disease stability for more than 6 months. Plasma AUCs on day 1 of successive weeks of treatment were stable over time. A nonsignificant decrease in serum insulin-like growth factor I levels was noted during treatment, but this trend was similar to that observed in three "control" patients treated with tamoxifen alone. When given with daily tamoxifen, the maximum tolerated dose of ATRA that could be given on alternate weeks was 190 mg/m2/day. This schedule of ATRA resulted in repeated periods of exposure to potentially therapeutic concentrations of ATRA. Declines in the serum insulin-like growth factor I concentrations observed in patients treated with tamoxifen and ATRA were similar to those observed in patients treated with tamoxifen alone. Objective responses were observed, some in patients who had previously progressed while receiving tamoxifen, suggesting that further studies would be of interest.

Delayed phospholipid degradation in rat brain after traumatic brain injury.

Lipid second messengers such as arachidonic acid and its metabolites and diacylglycerols (DAGs) are affected in brain injury. Therefore, changes in the pool size and the fatty acid composition of free fatty acids (FFAs) and DAGs were analyzed in different rat brain areas 4 and 35 days after traumatic injury. Cortical impact injury of low-grade severity was applied in the right frontal somatosensory cortex. Four days after injury, FFAs and DAGs were increased by three- and twofold, respectively, in the injured cortex and to a lesser extent in the contralateral cortex compared with sham-operated animals. Docosahexaenoic acid followed by stearic acid, and arachidonic acid, displayed the greatest changes in both FFAs and DAGs. By day 35, free stearic, oleic, and arachidonic acids remained elevated in the damaged cortex (1.5-fold each). DAGs showed the greatest change, reaching values 2.7-fold higher than sham in all frontal and occipital cortical areas, including brainstem. Oleoyl- and arachidonoyl-DAGs (four- and threefold increase, respectively) followed by docosahexaenoyl-DAGs (twofold) contributed to the DAG accumulation. These results reveal that traumatic brain injury triggers a sustained and time-dependent activation of phospholipase-mediated signaling pathways leading to membrane phospholipid degradation and targeting, early on, docosahexaenoyl phospholipid-enriched excitable membranes.

Immunoradiometric assay measurements of insulin-like growth factor-I (IGF-I): comparison with radioimmunoassays using native or des (1-3) IGF-I as radioligands.

Insulin-like growth-factor-binding proteins (BPs) in serum interfere with the measurement of insulin-like growth factor-I (IGF-I). Various assays have been developed to overcome this interference. We evaluated an immunoradiometric (IRMA) assay and compared it with the radioimmunoassay (RIA) using both native IGF-I and a truncated form of IGF-I [des (1-3) IGF-I] as radioligands. The IRMA was simpler (one step assay) and faster (3 hr incubation) than RIA(s) (overnight incubation). Sera were extracted with acid ethanol (AE) before all three assays. Analysis of serum samples (n = 78) performed by use of the two different radioligands in the RIA assays were highly correlated (r = 0.967, P < 0.0001). Measurements of serum IGF-I by IRMA in same samples were also highly correlated with those of the RIA assays (r = 0.952 for RIA and 0.947 for triGF-I RIA, P < 0.0001 for both). To assess the effect of binding protein-3 (BP-3) levels (after AE extraction) on these assays, BP-3 levels were measured in sera from 36 healthy women. The mean BP-3 level was 3.6 +/- 0.79 (S.D.) mg/L (range 1.3-5.0), and there was no significant difference in IGF-I levels measured by the three assays. Also, BP-3 levels were inversely correlated with IGF-I levels as measured by all three methods (r = 0.73 for IRMA, 0.71 for triGF-I, and 0.75 for IGF-I RIA). To assess the effect of binding protein-1 (BP-1) levels on these assays, IGF-I was also measured by IRMA and trlGF-I RIA in 19 women with advanced breast cancer. Women with breast cancer had significantly higher (P < 0.001) BP-1 levels than age matched healthy women. IGF-I levels measured by IGF-I IRMA were slightly lower than those measured by trlGF-I RIA in breast cancer patients. However, this difference was not statistically significant (P = 0.56). These findings suggest that variations in BP-3 or BP-1 levels after AE extraction have no significant effect in any of these assays. "We conclude that trlGF-I as a radioligand provides no added advantage over the standard IGF-I RIA. We also conclude that the IRMA assay is valid for measuring IGF-I and is faster and more convenient than RIA.

Nicotinic cholinergic receptors associated with mammalian cerebral vessels.

Current evidence suggests that the cerebral vasculature may be modulated by cholinergic nerves. We used ligand binding methods to examine the presence of nicotinic cholinergic receptors in brain vasculature. We found carbachol-displaceable [3H]acetylcholine (ACh) and [3H]nicotine (NIC) binding sites in preparations of intraparenchymal cerebral microvessels (CMV) and larger pial vessels from human and pig brains. Specific binding sites for [3H]ACh and [3H]NIC in cerebral microvessels were saturable and comparable in density to those in cerebral cortex. The Kds for the two ligands ranged 3-18 nM whereas the Bmaxs were 25-45 fmol/mg protein. In contrast, the binding of [3H]pirenzipine or [3H]quinuclidinyl benzilate, index for muscarinic receptors, was low (9-15% of cortex) in microvessels compared to the cerebral cortex. Our observations suggest the association of cholinergic nicotinic receptors with cerebral microvessels, which may be involved in the modulation of the cerebral circulation by cholinergic neurons.

Incorporation of trans long-chain n-3 polyunsaturated fatty acids in rat brain structures and retina.

During heat treatment, polyunsaturated fatty acids and specifically 18:3n-3 can undergo geometrical isomerization. In rat tissues, 18:3 delta 9c,12c,15t, one of the trans isomers of linolenic acid, can be desaturated and elongated to give trans isomers of eicosapentaenoic and docosahexaenoic acids. The present study was undertaken to determine whether such compounds are incorporated into brain structures that are rich in n-3 long-chain polyunsaturated fatty acids. Two fractions enriched in trans isomers of alpha-linolenic acid were prepared and fed to female adult rats during gestation and lactation. The pups were killed at weaning. Synaptosomes, brain microvessels and retina were shown to contain the highest levels (about 0.5% of total fatty acids) of the trans isomer of docosahexaenoic acid (22:6 delta 4c,7c,10c,13c,16c,19t). This compound was also observed in myelin and sciatic nerve, but to a lesser extent (0.1% of total fatty acids). However, the ratios of 22:6 trans to 22:6 cis were similar in all the tissues studied. When the diet was deficient in alpha-linolenic acid, the incorporation of trans isomers was apparently doubled. However, comparison of the ratios of trans 18:3n-3 to cis 18:3n-3 in the diet revealed that the cis n-3 fatty acids were more easily desaturated and elongated to 22:6n-3 than the corresponding trans n-3 fatty acids. An increase in 22:5n-6 was thus observed, as has previously been described in n-3 fatty acid deficiency. These results encourage further studies to determine whether or not incorporations of such trans isomers into tissues may have physiological implications.

Inhibitory effect of arachidonic acid on GTPase activating protein is antagonized by 1-stearoyl, 2-arachidonoyl glycerol.

The intrinsic GTPase activity of cellular protein p21ras is strongly increased by cytosolic GTPase activating protein (GAP). The activity of this enzyme has been shown to be inhibited by arachidonic acid. We report here that this inhibition is antagonized by the presence of 1-stearoyl, 2-arachidonoyl glycerol. This effect is structure specific and dose dependent, being maximum at 200 micrograms/ml of diacylglycerol (DG). These results suggest that production of DG in response to hormones or growth factors stimulation could indirectly modulate the interaction between p21ras and GAP.

Inhibitory effect of arachidonic acid on GTPase activating protein is antagonized by 1-stearoyl, 2-arachidonoyl glycerol.

The intrinsic GTPase activity of cellular protein p21ras is strongly increased by cytosolic GTPase activating protein (GAP). The activity of this enzyme has been shown to be inhibited by arachidonic acid. We report here that this inhibition is antagonized by the presence of 1-stearoyl, 2-arachidonoyl glycerol. This effect is structure specific and dose dependent, being maximum at 200 micrograms/ml of diacylglycerol (DG). These results suggest that production of DG in response to hormones or growth factors stimulation could indirectly modulate the interaction between p21ras and GAP.

Bradykinin receptors of cerebral microvessels stimulate phosphoinositide turnover.

We examined by ligand binding methods whether bradykinin (BK) receptors exist in rat and pig cerebral microvessels, and in the cerebral cortex from which the microvessels were isolated. We found a high-affinity and saturable BK receptor site in both rat and pig cerebral microvessels, but not in their cerebral cortex. The maximal density of binding and the dissociation constant were 8.0 +/- 4.1 and 6.8 +/- 1.5 fmol/mg of protein and 47 +/- 24 and 150 +/- 8 pM (mean +/- SD) in cerebral microvessels of the pig and rat, respectively. The high-affinity specific binding of BK was effectively displaced by des-Arg0[Hyp3-Thi5-8,D-Phe7]BK, a specific B2 receptor antagonist, but not by des-Arg9[Leu8]BK, a specific B1 antagonist. We also demonstrated that BK increases phosphatidylinositol hydrolysis in cerebral microvessels of the rat and pig. This effect was also blocked by the B2, but not by the B1, antagonist. Increased phosphatidylinositol hydrolysis was manifested by a rapid transient increase in inositol trisphosphate and the later slow accumulation of inositol bisphosphate and inositol monophosphate. Preincubation of microvessels with phorbol ester, stable GTP analogs, pertussis toxin, or in Ca(2+)-free buffer did not influence BK activation of phosphatidylinositol hydrolysis. These results demonstrate the existence of BK receptors of the B2 subtype in brain microvessels, which may play an important role in modulation of the brain microcirculation, probably via increased phosphoinositide turnover.

Effect of several vasoactive agents on guanylate cyclase activity in isolated rat brain microvessels.

We tested the ability of the following putative vasoactive agents to stimulate guanylate cyclase activity in isolated rat cerebral microvessels: angiotensin II, arginine vasopressin, atrial natriuretic peptide, bradykinin, carbachol and thrombin; at concentrations ranging between 10(-3) and 10(-9) M. The ability of cerebral microvessels to increase their cyclic GMP generation was ascertained in the presence of sodium nitroprusside. Of all the agents tested, only atrial natriuretic peptide stimulated cyclic GMP generation in isolated rat cerebral microvessels. Such stimulation was dose-dependent, reaching its maximum at 1 microM concentration. These results are consistent with the finding of atrial natriuretic peptide receptors in brain microvessels, and suggest that this peptide has an important role in modulating the function of brain capillaries, which constitute the blood-brain barrier. If receptors for the other vasoactive agents exist in brain microvessels, their action does not seem to be mediated by cyclic GMP as a second messenger.

Uptake and utilization of double-labeled high-density lipoprotein sphingomyelin in isolated brain capillaries of adult rats.

Isolated rat brain capillaries were incubated in the presence of high-density lipoprotein (HDL) containing [stearic acid-14C, (methyl-3H)choline]sphingomyelin. This double-labeled sphingomyelin was taken up in a concentration-dependent manner. Cerebral capillary-associated sphingomyelin had a 3H/14C ratio close to that of the incubation medium, a result indicating uptake of sphingomyelin without prior hydrolysis. TLC of lipid extracted from capillaries showed that part of the sphingomyelin (up to 40%) was hydrolyzed in the brain capillaries to ceramide and free fatty acids. The hydrolysis was proportional to the amount of incorporated sphingomyelin and reached a plateau when the HDL sphingomyelin concentration in the medium was 237 nmol/ml. The results of "pulse-chase" experiments showed that the choline moiety of sphingomyelin was recovered in the incubation medium after the chase period and that there was no redistribution of liberated choline in phosphatidyl-choline of capillaries.

Purified rat brain microvessels exhibit both acid and neutral sphingomyelinase activities.

Purified rat brain microvessels have been shown to hydrolyze radiolabeled sphingomyelin by means of two different enzyme systems. Enzymatic activity was detected at pH 7.4 and was strongly stimulated by magnesium or manganese and inhibited by calcium. Activity at pH 5.1 could also be found and was not dependent on any of these cations. At neutral pH and in the presence of magnesium, the rate of sphingomyelin hydrolysis did not exhibit a linear relationship with protein concentration. In contrast, increasing the protein concentration from 0.05 to 0.5 mg/ml resulted in a constant increase of sphingomyelin hydrolysis at pH 5.1. Kinetic parameters of both neutral and acid activities have been determined and were similar in magnitude to values reported previously for neural sphingomyelinases. This work demonstrates the occurrence of a neutral sphingomyelinase activity in purified rat brain microvessels, an observation raising the question of its role at the level of the blood-brain interface.

Alteration in fatty acid composition of adult rat brain capillaries and choroid plexus induced by a diet deficient in n-3 fatty acids: slow recovery after substitution with a nondeficient diet.

Wistar rats were fed for three generations with a semisynthetic diet containing either 1.5% sunflower oil (940 mg% of C18:2n-6, 6 mg% of C18:3n-3) or 1.9% soya oil (940 mg% of C18:2n-6, 130 mg% of C18:3n-3). At 60 days of age, the male offspring of the third generation were killed. The fatty acyl composition of isolated capillaries and choroid plexus was determined. The major changes noted in the fatty acid profile of isolated capillaries were a reduction (threefold) in the level of docosahexaenoic acid and, consequently, a fourfold increase in docosapentaenoic acid in sunflower oil-fed animals. The total percentage of polyunsaturated fatty acids was close to that in the soya oil-fed rats, but the ratio of n-3/n-6 fatty acids was reduced by threefold. In the choroid plexus, the C22:6n-3 content was also reduced, but by 2.6-fold, whereas the C22:5n-6 content was increased by 2.3-fold and the ratio of n-3/n-6 fatty acids was reduced by 2.4-fold. When the diet of sunflower oil-fed rats was replaced with a diet containing soya oil at 60 days of age, the recovery in content of n-6 and n-3 fatty acids started immediately after diet substitution; it progressed slowly to reach normal values after 2 months for C22:6n-5 and 2.5 months for C22:6n-3. The recovery in altered fatty acids of choroid plexus was also immediate and very fast. Recovery in content of C22:5n-6 and C22:6n-3 was complete by 46 days after diet substitution.

Acetoacetate, d-3-hydroxybutyrate and glucose utilization by capillaries isolated from developing rat brain.

Isolated cerebral capillaries from developing rats utilize glucose as well as ketone bodies essentially for oxidative metabolism. However, CO(2) production from [U-(14)C]glucose was significantly greater than from ketone bodies (except at 5 mM). Ketone body utilization (in the presence of 5 mM glucose in the incubation medium) was concentration-dependent (up to 5 mM). Lipid synthesis from ketone bodies was comparable to that from glucose up to 1 mM. At concentrations ? 1 mM, acetoacetate incorporation into total lipids and fatty acids was higher than other substrates, however, this difference was statistically significant only at 5 mM. Incorporation of substrates into sterols was very low (> 1 pmol/h/mg protein).

Ketone body utilization for energy production and lipid synthesis in isolated rat brain capillaries.

Isolated brain capillaries from 2-month-old rats were incubated for 2 h in the presence of [3-14C]acetoacetate, D-3-hydroxy[3-14C]butyrate, [U-14C]glucose, [1-14C]acetate or [1-14C]butyrate. Labelled CO2 was collected as an index of oxidative metabolism and incorporation of label precursors into lipids was determined. The rate of CO2 production from glucose was slightly higher than from the other substrates. Interestingly, acetoacetate was oxidized at nearly the same rate as glucose. This shows that ketone bodies could be used as a source of energy by brain capillaries. Radiolabelled substrates were also used for the synthesis of lipids, which was suppressed by the addition of albumin. The incorporation of [U-14C]glucose in total lipids was 10-times higher than that from other precursors. However, glucose labelled almost exclusively the glycerol backbone of phospholipids, especially of phosphatidylcholine. Ketone bodies as well as glucose were incorporated mainly into phospholipids, whereas acetate and butyrate were mainly incorporated into neutral lipids. The contribution to fatty acid synthesis of various substrates was in the following order: butyrate greater than or equal to acetate greater than ketone bodies greater than or equal to glucose. All precursors except glucose were used for sterol synthesis. Glucose produced almost exclusively the glycerol backbone of phospholipids.

Arachidonoyl-coenzyme A synthetase and nonspecific acyl-coenzyme A synthetase activities in purified rat brain microvessels.

Purified rat brain microvessels were prepared to demonstrate the occurrence of acyl-CoA (EC 6.2.1.3) synthesis activity in the microvasculature of rat brain. Both arachidonoyl-CoA and palmitoyl-CoA synthesis activities showed an absolute requirement for ATP and CoA. This activity was strongly enhanced by magnesium chloride and inhibited by EDTA. The apparent Km values for acyl-CoA synthesis by purified rat brain microvessels were 4.0 microM and 5.8 microM for palmitic acid and arachidonic acid, respectively. The apparent Vmax values were 1.0 and 1.5 nmol X min-1 X mg protein-1 for palmitic acid and arachidonic acid, respectively. Cross-competition experiments showed inhibition of radiolabelled arachidonoyl-CoA formation by 15 microM unlabelled arachidonic acid, with a Ki of 7.1 microM, as well as by unlabelled docosahexaenoic acid, with a Ki of 8.0 microM. Unlabelled palmitic acid and arachidic acid had no inhibitory effect on arachidonoyl-CoA synthesis. In comparison, radiolabelled palmitoyl-CoA formation was inhibited competitively by 15 microM unlabelled palmitic acid, with a Ki of 5.0 microM and to a much lesser extent by arachidonic acid (Ki, 23 microM). The Vmax of palmitoyl-CoA formation obtained on incubation in the presence of the latter fatty acids was not changed. Unlabelled arachidic acid and docosahexaenoic acid had no inhibitory effect on palmitoyl-CoA synthesis. Both arachidonoyl-CoA and palmitoyl-CoA synthesis activities were thermolabile. Arachidonoyl-CoA formation was inhibited by 75% after 7 min at 40 degrees C whereas a 3-min heating treatment was sufficient to produce the same relative inhibition of palmitoyl-CoA synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

The synthesis of lipids from [1-14C]acetate by isolated rat brain capillaries.

Lipid biosynthesis was investigated in isolated cerebral microvessels obtained from adult Sprague-Dawley rats using [1-14C]acetate as precursor. All lipid classes were labelled by [1-14C]acetate. Neutral lipids incorporated about 50% of radiolabelled acetate, among which free fatty acids and triglycerids showed the highest level of incorporation. Moreover, about 4% of radioactivity was found in cholesterol fraction. In phospholipid fraction, phosphatidylcholine and phosphatidylethanolamine were the main radiolabelled phospholipids. [1-14C]acetate was also incorporated into sulphatides and cerebrosides. The presence of bovine serum albumin in incubation medium modified the percentage of incorporation in different lipid fractions.