Hypothalamic delivery of doxycycline-inducible leptin gene allows for reversible transgene expression and physiological responses.

Our purpose was to incorporate regulation into the recombinant adeno-associated virus encoding leptin by introducing a tet-inducible promotor. This system, TET-Ob, allows for control of leptin gene expression via doxycycline in drinking water. F344XBN rats (aged 4 months) were given a hypothalamic injection of TET-Ob or control virus. During 34 days of doxycycline (doxy) administration to all rats (STAGE 1), TET-Ob rats gained 50.7% less mass, ate 10.4% less food, and had a 77.5% reduction in serum leptin as compared with controls. Doxy was then withdrawn from half of the TET-Ob rats for 32 days (TET-Ob-OFF), while half continued to receive doxy (TET-Ob-ON) (stage 2). During stage 2, TET-Ob-ON rats gained 44.8% less mass than TET-Ob-OFF and ate significantly less food than both TET-Ob-OFF and controls. Serum leptin increased to 83.4% of control values in TET-Ob-OFF, but remained very low in the in TET-Ob-ON. At death, visceral adiposity was 14.5% of controls in TET-Ob-ON animals, but had risen to 76.9% of controls in TET-Ob-OFF. A reversible increase in both leptin signal transduction in the hypothalamus and uncoupling protein expression in brown adipose was recorded. This system allows for more precise regulation of gene therapy-mediated fat loss.

Central leptin gene delivery evokes persistent leptin signal transduction in young and aged-obese rats but physiological responses become attenuated over time in aged-obese rats.

The purpose of this study was to determine if long-term leptin treatment desensitizes leptin signal transduction and the subsequent downstream anorexic and thermogenic responses in normal and leptin-resistant age-related obese rats. To this end, we administered, i.c.v., recombinant adeno-associated virus encoding rat leptin cDNA (rAAV-leptin) or control virus into young and aged-obese rats and after 9 or 46 days, examined food intake, oxygen consumption, body weight, serum leptin, STAT3 phosphorylation, hypothalamic NPY and POMC mRNAs, and UCP1 expression and protein level in brown adipose tissue (BAT). In young rats, rAAV-leptin depleted body fat and both anorexic and thermogenic mechanisms contributed to this effect. Moreover, leptin signal transduction was not desensitized, and there were persistent physiological responses. Similarly, in the aged-obese rats, there was unabated leptin signal transduction, however, both the anorexic and thermogenic responses completely attenuated sometime after day 9. This attenuation, downstream of the leptin receptor, may be contributing to the leptin-resistance and age-related weight gain in these aged-obese rats. Finally, in young rats, although the initial responses to rAAV-leptin were dominated by anorexic responses, by 46 days, the predominant response was thermogenic rather than anorexic, suggesting that energy expenditure may be an important component of long-term weight maintenance.

Resistance to the anorexic and thermogenic effects of centrally administrated leptin in obese aged rats.

The aim of the present study was to determine whether the anorexic and thermogenic effects of leptin were attenuated in overweight aged rats following intracerebroventricular (i.c.v.) injection of murine leptin. Male F344/BN rats of two ages (6 months: young (n=20) and 24 months: old (n=18)) were divided into three groups (control, pair-fed and leptin) and were treated with either vehicle (artificial cerebrospinal fluid) or leptin (15.6 microgram/day) for 3 days. There was an age-related increase in basal food intake (20+/-2%), serum leptin levels (363+/-106%) and leptin (OB) mRNA (72+/-16%) in perirenal white adipose tissue (PWAT). In contrast, basal expression of hypothalamic NPY mRNA and brown adipose tissue (BAT) uncoupling protein 1 (UCP1) mRNA was reduced significantly (-35+/-4% and -51+/-5%, respectively) with age. I.c.v. leptin treatment had a significantly greater effect in reducing food intake (-42+/-5% vs. -23+/-4%), serum leptin levels (-55+/-7% vs. 10+/-2%) and PWAT OB mRNA (-46+/-2% vs. 10+/-5%) in young than in old rats. Similarly, central leptin treatment also had a greater effect in suppressing hypothalamic NPY mRNA expression in young (-23+/-4%) than in old (-8+/-4%) rats compared with their age-matched pair-fed treated rats. The stimulatory effect of i.c.v. leptin treatment on BAT UCP1 mRNA expression was also significantly greater in young rats (45+/-8%) than in old rats (10+/-6%) compared with age-matched pair-fed rats. Our previous report indicated that these overweight aged rats were resistant to peripheral administered leptin. The present data extend those findings and demonstrate that the impaired anorexic and metabolic effects of leptin are centrally mediated. This leptin resistance may be due to either the elevated obesity and serum leptin with age or due to age itself or both. The development of leptin resistance with age may contribute to the hyperphagia, hyperleptinemia and impaired energy balance with age.

Impaired leptin signal transduction with age-related obesity.

Leptin contributes to the regulation of both food intake and energy expenditure. We previously demonstrated that the F-344xBN rat, a rodent model for late-onset obesity, is leptin resistant, suggesting that leptin signal transduction may be impaired in these aged, overweight rats. To test this hypothesis, we examined the in vivo dose-response and time-course response of leptin-induced STAT3 activation (phosphorylation and binding activity to the SIE M67 oligonucleotide) in the hypothalamus of young rats along with the dose-response leptin-induced STAT3 phosphorylation (P-STAT3) and maximum increase in binding activity in young and aged rats. In young rats there was a dose (0-1 mg, iv) and time dependent increase in P-STAT3 and in P-STAT3 binding activity. P-STAT3 paralleled the rise and fall in serum leptin levels with P-STAT3 elevated for at least 4 h with return to basal levels by 14 h after 1 mg leptin. The maximum level of leptin-induced P-STAT3 was unchanged with age, but the dose for half maximal phosphorylation was greater in aged (138 microg) compared with young (26 microg) rats. In addition, the leptin-induced increase in P-STAT3 transcription factor binding was diminished in aged rats. These data suggest that leptin signal transduction, in vivo, demonstrate a time and dose response increase paralleling the rise and fall in serum leptin, suggesting that serum leptin levels are the most important factor in determining leptin-induced phosphorylation of STAT3 in the hypothalamus. In addition, aged, overweight rats demonstrate reduced signal transduction in response to leptin, with reduced sensitivity for STAT3 phosphorylation and diminished leptin-induced P-STAT3 transcription factor binding. This impaired leptin signal transduction may be due to either the elevated obesity with age or due to age itself or both.

Hyperhomocyst(e)inemia induces multiorgan damage.

Hyperhomocyst(e)inemia has been associated with the development of hypertension, stroke, and cardiovascular, cerebral/neuronal, renal, and liver diseases. To test the hypothesis that homocyst(e)ine plays an integrated role in multiorgan injury in hypertension, we employed: (1) spontaneously hypertensive rats (SHR) in which endogenous homocyst(e)ine levels are moderately high (18.1 +/- ().5 microM); (2) control age- and sex-matched Wistar Kyoto (WKY) rats in which homocyst(e)ine levels are normal (3.7 +/- 0.3 microM). To create the pathophysiological condition of hyperhomocyst(e)inemia, 20 mg/day homocyst(e)ine was administered for 12 weeks in (3) SHR (SHR-H) and in (4) WKY (WKY-H) rats. (5) Endogenous homocyst(e)ine levels were reduced slightly but not significantly from 18.1 +/- 0.5 microM to 12.5 +/- 0.7 microM in SHR by folic acid administration (SHR-F). Plasma and tissue levels of homocyst(e)ine were determined by HPLC and spectrophotometric methods. Plasma and sympathetic ganglion (neuronal) matrix metalloproteinase (MMP) activity was measured by zymography. Activity of neuronal MMP was increased in hyperhomocyst(e)inemic rats as compared with controls. Mean arterial pressure (mmHg) was 95 +/- 5, 126 +/- 8,157 +/- 10, 188 +/- 5, and 165 +/- 12 in WKY, WKY-H, SHR, SHR-H, and SHR-F, respectively. Urinary protein (mg/day) was 0.11 +/- 0.03, 0.88 +/- 0.22, 0.47 +/- 0.10, 0.89 +/- 0.21, and 0.81 +/- 0.21 in WKY, WKY-H. SHR, SHR-H, and SHR-F, respectively, as measured by the Bio-Rad dye binding assay. The relationships between increased arterial pressure, plasma homocyst(e)ine, and urinary protein were delineated. Plasma and neuronal creatinine phosphokinase (CK) isoenzymes were measured by agarose gel electrophoresis. All three CK isoenzymes, i.e., MM, MB, and BB, specific for skeletal, cardiac, and nerve tissue, respectively, were induced following 12 weeks' hyperhomocyst(e)inemia, suggesting multiorgan injury by homocyst(e)ine. Homocyst(e)ine induces endocardial endothelial cell (capillary) apoptosis and may reduce capillary cell density. Structural damage to aorta, myocardium, kidney, and renalureter was analyzed by histology. Results suggested an integrated physiological role of homocyst(e)ine in injury to the endothelial/epithelial cell lining in the respective organs.

Sodium induces hypertrophy of cultured myocardial myoblasts and vascular smooth muscle cells.

The mechanisms of sodium-induced myocardial hypertrophy and vascular hypertrophy are poorly understood. We tested the hypothesis that a high sodium concentration can directly induce cellular hypertrophy. Neonatal rat myocardial myoblasts (MMbs) and vascular smooth muscle cells (VSMCs) were cultured in a 50:50 mixture of DMEM and M199 supplemented with 10% fetal bovine serum. When the monolayers reached approximately 80% confluence, normal sodium medium (146 mmol/L) was replaced with high sodium media (152 mmol/L, 160 mmol/L, and 182 mmol/L) for up to 5 days. Increasing sodium from a baseline concentration of 146 mmol/L to the higher concentrations for 5 days caused dose-related increases in cell mean diameter, cell volume, and cellular protein content in both MMbs and VSMCs. Increasing the sodium concentration by only 4% (from 146 mmol/L to 152 mmol/L) caused the following respective changes in MMbs and VSMCs: 8.5% and 8.7% increase in cell mean diameter, 27.6% and 27.0% increase in cell volume, and 55.7% and 46.7% increase in cellular protein content. The rate of protein synthesis, expressed as [3H]leucine incorporation, increased by 87% and 99% in MMbs after exposure to 152 mmol/L and 160 mmol/L sodium, respectively, compared with the 146-mmol/L sodium control group. Exposure of MMbs to medium with a sodium concentration of 10% above normal, ie, 160 mmol/L, caused a significant decrease (range, 26% to 44%) in the rate of protein degradation at multiple time points over a 48-hour period compared with normal sodium control cells. The increase in cellular protein content caused by 160 mmol/L sodium returned to normal within 3 days after MMbs were returned to a normal sodium medium. These findings support the hypothesis that sodium has a direct effect to induce cellular hypertrophy and may therefore be an important determinant in causing myocardial and/or vascular hypertrophy in subjects with increased sodium concentration in the extracellular fluid.

Abnormal kidney function as a cause and a consequence of obesity hypertension.

1. Obesity is the most common nutritional disorder in the US and is a major cause of human essential hypertension. Although the precise mechanisms by which obesity raises blood pressure (BP) are not fully understood, there is clear evidence that abnormal kidney function plays a key role in obesity hypertension. 2. Obesity increases tubular reabsorption and this shifts pressure natriuresis towards higher BP. The increased tubular reabsorption is not directly related to hyperinsulinaemia, but is closely linked to activation of the sympathetic and renin-angiotensin systems, and possible changes in intrarenal physical forces caused by medullary compression due to accumulation of adipose tissue around the kidney and increased extracellular matrix within the kidney. 3. Obesity is also associated with marked renal vasodilation and increased glomerular filtration rate, which are compensatory responses that help overcome the increased tubular reabsorption and maintain sodium balance. However, chronic renal vasodilation causes increased hydrostatic pressure and wall stress in the glomeruli which, along with increased lipids and glucose intolerance, may cause glomerulosclerosis and loss of nephron function in obese subjects. Because obesity is a primary cause of essential hypertension as well as type II diabetes, there is good reason to believe that obesity may also be the most frequent cause of end-stage renal disease. 4. Future research is needed to determine the mechanisms by which excess weight gain activates the neurohumoral systems and alters renal structure and function. Because of the high prevalence of obesity in most industrialized countries, unravelling these mechanisms will likely provide a better understanding of the pathophysiology of human essential hypertension and chronic renal failure.

Chronic leptin infusion increases arterial pressure.

Plasma leptin concentration is increased in hypertensive obese humans, but whether leptin contributes to the increased arterial pressure in obesity is not known. In this study, we tested whether chronic increases in leptin, to levels comparable to those in obesity, could cause a sustained increase in arterial pressure and also the importance of central nervous system (CNS) versus systemic mechanisms. Five male Sprague-Dawley rats were implanted with chronic nonoccluding catheters in the abdominal aorta and both carotid arteries for CNS infusion, and five other rats were implanted with an abdominal aorta catheter and femoral vein catheter for intravenous (I.V.) infusion. After 7 days of control, leptin was infused into the carotid arteries or femoral vein at 0.1 microg/kg/min for 5 days and 1.0 microg/kg/min for 7 days, followed by a 7-day recovery period. The carotid artery and i.v. infusions of leptin at 1 microg/kg/min significantly increased plasma leptin levels, from 1.2+/-0.4 ng/mL to 91+/-5 ng/mL and from 0.9+/-0.1 ng/mL to 94+/-9 ng/mL, respectively, but there was no significant increase in either group at the low dose. Food intake also did not change at the low dose but decreased by approximately 65% in the carotid group and 69% in the i.v. group after 7 days of the 1 microg/kg/min infusion. Mean arterial pressure (MAP) increased slightly at the low dose only in the carotid group, but this was not statistically significant. At the higher dose, however, MAP increased significantly from 86+/-1 mm Hg to 94+/-1 mm Hg in the carotid group and from 87+/-1 mm Hg to 93+/-1 mm Hg in the i.v. group. Heart rate also increased significantly in both groups at 1 microg/kg/min leptin infusion. Fasting blood glucose and insulin levels decreased significantly at 1 microg/kg/min in both the carotid artery group (-10.5% and -82.5%, respectively) and the i.v. group (-13.6% and -80.4%, respectively). All variables returned to control levels after leptin infusion was stopped. These results indicate that chronic increases in circulating leptin cause sustained increases in arterial pressure and heart rate and are consistent with a possible role for leptin in obesity hypertension.

Prodrug esters of the indolocarbazole CEP-751 (KT-6587).

Prodrug esters of the indolocarbazole CEP-751 (KT-6587) were prepared with the goal of identifying water soluble, stable but cleavable forms for intravenous dosing. A dipeptide proform Lys-beta-Ala (16, CEP-2563/KT-8391) was identified for advancement to clinical trials.

Inhibition of thromboxane synthesis attenuates insulin hypertension in rats.

Chronic insulin infusion in rats increases mean arterial pressure (MAP) and reduces glomerular filtration rate (GFR), but the mechanisms for these actions are not known. This study tested whether thromboxane synthesis inhibition (TSI) would attenuate the renal and blood pressure responses to sustained hyperinsulinemia. Male Sprague-Dawley rats were instrumented with arterial and venous catheters, and MAP was measured 24 h/day. After 4 days of baseline measurements, endogenous synthesis of thromboxane was suppressed in 7 rats by infusing the thromboxane synthetase inhibitor, U63557A, intravenously (30 microg/kg/min) for the remainder of the experiment; 7 other rats received vehicle. Baseline MAP was not significantly different between vehicle and TSI rats (96 +/- 1 v 99 +/- 1 mm Hg). After 3 days of U63557A or vehicle, a 5-day control period was started, followed by a 7-day infusion of insulin (1.5 mU/kg/min, intravenously). Glucose (22 mg/kg/min, intravenously) was infused along with insulin to prevent hypoglycemia. In the control period, MAP was not different between vehicle and TSI rats (99 +/- 2 v 100 +/- 1 mm Hg), but MAP increased throughout the 7-day infusion period only in the vehicle rats with an average increase in blood pressure of 7 +/- 2 mm Hg. In the control period, GFR was lower in vehicle rats compared with TSI rats (2.5 +/- 0.1 v 3.1 +/- 0.2 mL/min, P = .06), and the decrease to 81% +/- 4% and 91% +/- 6% of control, respectively, during insulin was significant only in the vehicle rats. All variables returned toward control during a 6-day recovery period. These results suggest that full expression of hypertension and renal vasoconstriction during hyperinsulinemia in rats is dependent on a normal ability to synthesize thromboxane.

Insulin-induced hypertension in rats depends on an intact renin-angiotensin system.

This study tested the dependence of insulin-induced hypertension in rats on a functional renin-angiotensin system. Rats were instrumented with chronic artery and vein catheters and housed in metabolic cages. After acclimation, 10 rats began receiving the angiotensin-converting enzyme inhibitor (ACEI) benazepril at 1.8 mg.kg-1.d-1 via a continuous intravenous infusion that was maintained throughout the study; 8 control rats received vehicle. Four days after starting ACEI or vehicle, all rats entered a 5-day control period that was followed by a 7-day insulin infusion at 1.5 mU.kg-1.min-1. Glucose was coinfused at 22 mg.kg-1.min-1 to prevent hypoglycemia. Insulin infusion in control rats increased mean arterial pressure (MAP; measured 24 h/d) from an average of 101 +/- 1 to 113 +/- 2 mm Hg on day 1; MAP averaged 110 +/- 1 mm Hg for the 7-day infusion period. Glomerular filtration rate decreased, although not significantly, from 2.7 +/- 0.1 to 2.1 +/- 0.2 mL/min on day 3. Chronic ACEI decreased baseline MAP from an average of 97 +/- 1 to 79 +/- 1 mm Hg and markedly attenuated the increase in MAP during insulin. MAP averaged 81 +/- 1 mm Hg for the 7-day period and increased significantly, to 85 +/- 2 mm Hg, only on day 3. Likewise, the tendency for glomerular filtration rate to decrease was blunted. These results indicate that insulin-induced hypertension in rats depends on angiotensin II and suggest that a reduction in glomerular filtration rate contributes to the shift in pressure natriuresis.

Thromboxane is required for full expression of angiotensin hypertension in rats.

Recent studies suggest that thromboxane (TX) mediates a significant component of angiotensin II (ANG II)-induced hypertension. However, there is little information to support the hypothesis that this relationship is important during chronic, physiological increases in ANG II, particularly while controlling for variation in endogenous ANG II levels induced by TX inhibition. This study tested that hypothesis in 27 chronically instrumented rats. After baseline measurements, suppression of endogenous TX was induced and maintained throughout the study in 13 rats by i.v. infusion of the TX synthesis inhibitor (TSI) U63557A: the other 14 rats received vehicle. Baseline mean arterial pressure (MAP) was not different between groups and was unchanged by TSI or vehicle. Continuous inhibition of ANG II production was then initiated in both groups of rats by i.v. infusion of the angiotensin-converting enzyme inhibitor (ACEI) benazepril. ACEI reduced blood pressure similarly in vehicle and TSI rats, from 105 +/- 2 to 91 +/- 2 mm Hg and 103 +/- 1 to 89 +/- 1 mm Hg, respectively. ANG II was then infused at 5 ng.kg-1.min-1 i.v. for 7 days in six rats from each group to restore ANG II activity to baseline levels. This dose increased MAP to 103 +/- 2 and 101 +/- 1 mm Hg in vehicle and TSI rats, respectively, values not different from pre-ACEI levels. Seven TSI rats and eight vehicle rats received a higher dose of ANG II (20 ng.kg-1.min-1 i.v.). After 7 days, MAP was higher in vehicle than in TSI rats (143 +/- 5 versus 120 +/- 4 mm Hg). These results suggest that endogenous TX is an important determinant of MAP in ANG II hypertension but may have a diminished role in blood pressure regulation when ANG II is at normal and subnormal levels.

Central role of the kidney and abnormal fluid volume control in hypertension.

In human essential hypertension, and in all forms of experimental hypertension studied thus far, volume regulation and the relationship between blood pressure (BP) and sodium excretion (pressure natriuresis) are abnormal. Considerable evidence indicates that resetting of pressure natriuresis plays a key role in causing hypertension, rather than merely occurring as a consequence of increased BP. In patients with essential hypertension, resetting of pressure natriuresis is characterized either by a parallel shift to higher BPs and salt-insensitive hypertension, or by a decreased slope of pressure natriuresis and salt-sensitive hypertension. This clearly indicates that essential hypertension cannot be ascribed to a single abnormality of kidney function. Multiple physiological studies have shown that salt-sensitive hypertension can be elicited by renal abnormalities that cause either loss of functional kidney mass or an inability to modulate the renin-angiotensin-aldosterone (RAA) system appropriately; these abnormalities include loss of functional nephrons, decreased glomerular capillary filtration coefficient, patchy renal ischemia, and increased distal and collecting tubular reabsorption. Renal abnormalities that cause salt-insensitive hypertension are characterized by normal functional kidney mass, and the ability to appropriately modulate the renin-angiotensin system during changes in sodium intake; important causes of salt-insensitive hypertension include widespread increases in preglomerular resistance and increased reabsorption in the proximal tubules and loops of Henle. By comparing the characteristics of pressure natriuresis in essential hypertensive subjects with those found in experimental hypertension of known origin, we can gain considerable insight into the etiology of human hypertension.

Effects of ATP-sensitive K(+)-channel activators on transmitter release parameters at the frog neuromuscular junction.

The frog neuromuscular junction was used to study KATP channel action and its relation to transmitter release. Diazoxide (10 microM) and cromakalim (300 microM) decreased the amplitude of the end-plate potential (EPP) without significantly affecting the miniature end plate potential (MEPP) amplitude. Thus, there was a significant decrease in the quantal content of EPP after administration of the KATP channel activators. These agents did not alter MEPP frequency or resting membrane potential (RMP). The diazoxide-induced decrease in EPP amplitude was antagonized by the KATP blocker glibenclamide (10 microM) suggesting KATP channel involvement. Glibenclamide (10 microM) by itself caused a significant decrease in the RMP without significantly affecting the parameters of transmitter release. The diazoxide-induced decrease in EPP amplitude was not readily reversible with washing, however, when glibenclamide was added after diazoxide, the effect was easier to reverse. The results indicate that the activators exert their effect predominantly at the presynaptic region.

Brain and CSF specific chemical delivery systems for beta-lactam antibiotics. Study of two dihydropyridine derivatives of benzylpenicillin in rabbits and dogs.

Following previous studies in rats, the ability of two chemical delivery systems (CDSs) to deliver benzyl penicillin (1) to the central nervous system of rabbits and dogs was investigated. One of the systems (3) was a diester of methylene diol, and the other (5) a diester of ethylene 1,2-diol; in both, one hydroxyl group of the diol was esterified by the 3-carboxylic acid group of benzylpenicillin, and the other by the carboxy group of an N-methyldihydropyridine (dihydrotrigonelline). The basis of the system is the ability of the dihydropyridine components to undergo oxidation to quaternary pyridinium salts (2 from 3, and 4 from 5). In vitro relative stability studies were first performed in 10% rabbit brain homogenate, rabbit CSF and dog CSF. The results showed that the CDSs (3 and 5) were more stable than the corresponding quaternary salts (2 and 4). Hydrolysis of 2 and 3 resulted in the release of 1, whereas hydrolysis of 4 and 5 released both 1 and the hydroxyethyl ester (6) of 1. In vivo distribution studies were performed in rabbits and dogs. After i.v. administration of equimolar doses of 1 or the CDSs, levels of 1 in brain and CSF were substantially higher and more prolonged in the cases of the CDSs than in the case of 1 itself. Brain levels of 1 were lower following administration of 5, as compared with 3, due to the release of the intermediate compound, the hydroxyethyl ester (6) of 1, which was not hydrolyzed efficiently to 1 in rabbit or dog brain. The substantially increased and prolonged penicillin levels following administration of the CDSs arise as the result of improved penetration of the lipophilic CDSs across the blood-brain barrier, and a "lock-in" effect of the corresponding quaternary salts generated in situ.

Chemical delivery systems for drugs containing an amino group: synthesis and properties of some pyridine derivatives of desipramine.

Seven chemical delivery systems (CDS) based on a dihydropyridine<-->quaternary pyridinium salt type redox system and analogous to the naturally occurring NADH<-->NAD+ coenzyme system were applied in the case of the antidepressant drug desipramine. The pyridine moiety-containing carriers were linked to the amino function of desipramine either as amides or substituted carbamates. Lipophilic properties were expressed in terms of chromatographic Rm values. Oxidative stability of the dihydropyridine forms of the CDSs were determined in vitro. The amide type derivatives were stable toward hydrolysis in buffers and in biological fluids, whereas the carbamates released the parent drug in a very efficient manner. In a behavioral despair test, the CDSs did not show improved activity when compared to desipramine. In vivo distribution studies of one of the CDS did not show more efficient delivery of the desipramine into the rat brain but did show a prolonged presence at a constant level.

Improved anticonvulsant activity of phenytoin by a redox brain delivery system. III: Brain uptake and pharmacological effects.

Phenytoin (DPH) was delivered to the brain by a dihydropyridine in equilibrium pyridinium salt redox system, which was evaluated for anticonvulsant activity. Following iv injection of the lipophilic delivery system of DPH (2) to rats, concentrations of DPH were lower but sustained and, after 30 min, essentially the same as the levels after equimolar administration of DPH. While 2 delivered the same levels of DPH to the brain as DPH did, it was twice as potent as DPH in rats (ED50 was 7.5 mumol/kg for 2 and 14.2 mumol/kg for DPH) and mice (2: 10.5; DPH: 23.9) against maximal electroshock seizures (MES), and seven times more potent in mice (2: 10.0, DPH: 70.6) against maximal pentylenetetrazole seizures (MPS). Moreover, 2 was active against pentylenetetrazole threshold seizures (PTS) in mice and rats (ED50 = 44.1 and 40.5 mumol/kg, respectively), while DPH was ineffective (up to a dose of 79.2 mumol/kg). After evaluation of acute neurological toxicity in rats, 2 was found to possess 1.5 times higher a protective index (for MES) than DPH. It appeared also that while DPH was 2.9 times less sensitive to MPS than to MES, 2 was equally potent to both types of convulsions. Thus, the data indicate that 2 delivered DPH more efficiently to the brain. The better anticonvulsant activity (quantitatively as well as qualitatively) of 2 can be explained on the basis of an improved distribution in the brain due to its higher lipophilicity, and by favorable regional differences in the rates of conversion of 2 to DPH at the convulsing foci.

Improved anticonvulsant activity of phenytoin by a redox brain delivery system. II: Stability in buffers and biological materials.

The stability of nine chemical delivery systems (CDSs) for phenytoin (DPH) was studied in aqueous buffers and in biological materials. The systems were based on a dihydropyridine in equilibrium quaternary pyridinium salt redox pair attached to 3-(hydroxymethyl)phenytoin via an ester linkage. The pyridinium derivatives released DPH in aqueous buffers and their hydrolytic reactivity was consistent with their chemical structure. Although in rat blood and plasma all pyridinium esters hydrolyzed rapidly, there was a wide range in the hydrolysis rates in rat brain homogenate. The sterically hindered 1-alkylcarboxynicotinamide was the least reactive ester (t1/2 = 98.2 min), while the trigonellylglycolate ester was the fastest to hydrolyze enzymatically (t1/2 = 2 min) in rat brain homogenate. In acidic media, the major products of all dihydropyridine esters were the corresponding water adducts, the 6-hydroxy- 1,4,5,6-tetrahydropyridines. These adducts were of no significance in biological materials. After comparison of the relative stability of the corresponding pairs of dihydropyridine and pyridinium ion in brain homogenate and the absolute stability of the various dihydropyridines, two CDSs were chosen for further in vivo evaluations. The CDSs chosen were the dihydrotrigonellinate ester and its 6-methyl derivative.

Improved anticonvulsant activity of phenytoin by a redox brain delivery system I: Synthesis and some properties of the dihydropyridine derivatives.

Nine chemical delivery systems (CDSs) were synthesized for the efficient transport of phenytoin (DPH) across the blood-brain barrier. The CDSs were based on a dihydropyridine in equilibrium quaternary pyridinium ion redox system which relies on chemistry similar to the NADH in equilibrium NAD interconversion for activity. The chemical carriers, derivatives of trigonelline, 1-alkylcarboxynicotinamide, 3-pyridylacetic acid, and N-methylpicolinic acid, were esterified with 3-(hydroxymethyl)phenytoin. The CDSs proved to be more lipophilic (5-23 times) than DPH. The 1-alkylcarboxydihydronicotinamide CDSs, excluding the sterically hindered one (11e), were quite unstable in rat tissue homogenates and hydrolyzed to release DPH. In human blood, however, they were found to be much more stable (75 times) toward hydrolysis. All other CDSs were oxidized quantitatively to the corresponding pyridinium ion in rat brain homogenates. These compounds were found to possess the required physicochemical characteristics for delivering DPH into rat brain.