Donna Wielbo

Antisense inhibition of AT1 receptor mRNA and angiotensinogen mRNA in the brain of spontaneously hypertensive rats reduces hypertension of neurogenic origin

To determine the role of angiotensinogen and angiotensin II type-1 (AT1) receptor genes in hypertension, spontaneously hypertensive rats (SHR) were injected with synthetic antisense oligodeoxynucleotides (ODNs), intracerebroventricularly (i.c.v). Antisense ODNs were constructed to bases -5 to +13 of angiotensinogen mRNA (18-mer) and to bases +63 to +77 (15-mer) of angiotensin II type-1 receptor mRNA. Hypertension was significantly reduced by the application of 50 micrograms of both antisense ODNs to normotensive levels. The phosphorothioated antisense ODN to the AT1 receptor produced long-lasting (7 days) decreases in blood pressure. After AT1 antisense treatment, AT1 receptors were reduced in the paraventricular nucleus (PVN) and in the anterior third ventricle area (AV3V). Following angiotensinogen antisense treatment, angiotensin II levels were significantly reduced in the brainstem (P < 0.05), indicating arrest of angiotensin II synthesis. The results demonstrate that inhibiting the brain renin-angiotensin system by antisense inhibition of the angiotensinogen and the AT1 receptor genes, lowers high blood pressure in the SHR. The antisense administration to specific genes of the tissue renin-angiotensin system offers the possibility of a new approach to developing antihypertension treatments.

Antisense Inhibition of Hypertension in the Spontaneously Hypertensive Rat

Phosphorothioated antisense oligodeoxynucleotide (ASODN) targeted to angiotensinogen mRNA was administered intracerebroventricularly in spontaneously hypertensive rats to test whether angiotensinogen reduction would lower their hypertensive blood pressures. The ASODN lowers hypertensive blood pressures to normotensive levels in spontaneously hypertensive rats; sense oligodeoxynucleotide had no effect. Administration of phosphorothioated ASODN produced a prolonged duration of lowered blood pressure. Injections of ASODN at the same dose that decreased hypertension when administered centrally did not result in blood pressure decreases when administered intra-arterially. Furthermore, angiotensinogen production was decreased in the brain stem and significantly decreased in the hypothalamus of the ASODN-treated rats (P < .05), supporting the concept of centrally mediated regulation of hypertension by an overactive brain angiotensin system. To determine the distribution of centrally administered oligodeoxynucleotides, fluorescein isothiocyanate-conjugated oligodeoxynucleotides were injected directly into the lateral ventricles. One hour later, oligodeoxynucleotides were distributed throughout the lateral and third ventricles, with tissue and cellular uptake observed in discrete cells at the injection site. This indicates that the oligodeoxynucleotides are taken up rapidly by brain cells and that they permeate the areas surrounding brain nuclei involved in central blood pressure regulation and volume homeostasis. The results confirm and extend our previous study with phosphodiester ASODN and show that phosphorothioation modification increases the duration of the response and is taken up in vivo. We conclude that with modification, ASODN inhibition of angiotensinogen mRNA translation can be used for a prolonged, profound decrease in mean arterial pressure in the spontaneously hypertensive rat through a central mechanism.

Inhibition of Hypertension by Peripheral Administration of Antisense Oligodeoxynucleotides

We administered liposome-encapsulated antisense oligodeoxynucleotide targeted to angiotensinogen mRNA peripherally to spontaneously hypertensive rats to test whether peripheral angiotensinogen reduction would lower their hypertensive blood pressures and to determine the role of peripheral angiotensinogen in the modulation of hypertension. Using in vitro translation techniques, we tested the sequence specificity of the antisense sequence. The selected antisense sequence decreased angiotensinogen production in vitro, enabling us to distinguish between specific and nonspecific effects. To increase the efficiency of peripheral and hepatic antisense delivery, oligonucleotides were liposome encapsulated and intra-arterial administration. Confocal microscopy was used for determination of the hepatic distribution of fluorescently labeled antisense. Encapsulated antisense molecules were seen to be distributed within liver tissue 1 hour after injection; however, little or no uptake was observed with the unencapsulated oligonucleotides. We also determined the physiological effects of antisense oligodeoxynucleotide targeted to liver angiotensinogen mRNA. Administration of liposome-encapsulated antisense significantly decreased hypertensive blood pressures to normotensive levels compared with scrambled control oligonucleotides, unencapsulated antisense, and empty liposomes (P = .013). These data were supported by biochemical changes elicited by the antisense treatment. Rats receiving liposome-encapsulated antisense had significantly lowered peripheral angiotensinogen and angiotensin II levels compared with control groups (P < .05). No significant heart rate changes were observed in the antisense or control groups. These results suggest that peripheral angiotensinogen plays a role in the maintenance of hypertensive blood pressure in this model of hypertension and that peripheral administration of antisense molecules is possible with organ-targeted delivery mechanisms.

Antisense oligonucleotide to AT1 receptor mRNA inhibits central angiotensin induced thirst and vasopressin

Antisense oligodeoxynucleotides (AS-ODN) to AT1 receptor mRNA inhibit high blood pressure in Spontaneously Hypertensive Rats (SHR) when injected into the brain. The effect is presumably through inhibition of the actions of brain angiotensin II (Ang II). Central injection of Ang II elicits several physiological responses including release of vasopressin and motivation to drink. The angiotensin II type-I (AT1) receptor is located in brain regions which have been implicated in mediating these effects. Therefore we hypothesized that AS-ODN to AT1 mRNA would inhibit the drinking and AVP response to central administration of Ang II in adult male SHR. AS-ODN were constructed to bases +63 to +77 (15-mer) of the AT1 receptor RNA. 24 h after AS-ODN treatment (50 micrograms/4 microliters) (intracerebroventricularly, i.c.v.), the drinking response to Ang II (50 ng, i.c.v.) was significantly reduced in the SHR (P < 0.05). The drinking response to Ang II (i.c.v.) was also reduced in the Sprague-Dawley rats (P < 0.05). There was no reduction of water intake in the control animals treated with scrambled ODN (SC-ODN). Repeated injection of AS-ODN did not produce a greater reduction in drinking response. Arginine vasopressin (AVP) release to central Ang II was significantly decreased after AS-ODN treatment when compared to vehicle (P < 0.05) and to SC-ODN injections (P < 0.05). Radioligand binding assays of the hypothalamic block after AS-ODN treatment showed a significant decrease of AT1 receptor binding (P < 0.05). The results show that the antisense inhibition of brain AT1 receptor gene expression decreases the Ang II induced drinking and AVP release responses.

Analgesic effects of codeine-6-glucuronide after intravenous administration

Centrally administered codeine glucuronide has been shown to exhibit antinociceptive properties with decreased immunosuppressive effects compared to codeine. In this study, codeine‐6‐glucuronide was administered to rats, and its analgesic effect was compared to that of codeine. The concentrations of codeine and its metabolites in plasma and brain were also determined at the peak response time after administration of each compound. Receptor‐binding studies with rat brain homogenates and affinity profiles were also determined. Intravenous administration of codeine‐6‐glucuronide resulted in approximately 60% of the analgesic response elicited by codeine itself. Analysis of plasma and brain showed that codeine‐6‐glucuronide is relatively stable in vivo, with only small amounts of morphine‐6‐glucuronide being detected in addition to unchanged codeine‐6‐glucuronide. The receptor affinity of codeine‐6‐glucuronide was similar to that of codeine. It is concluded that intravenously administered codeine‐6‐glucuronide possesses analgesic activity similar to that of codeine, and may have clinical benefit in the treatment of pain

Analgesic and immunomodulatory effects of codeine and codeine 6-glucuronide

AbstractPurpose. The antinociceptive and immunosuppressive effects of codeine and codeine 6-glucuronide were determined in rats after intra-cerebroventricular administration. Methods. Codeine 6-glucuronide was synthesized using a modification of the Koenigs-Knorr reaction. A lipophilic intermediate formed during synthesis, methyl [codein-6-yl-2,3,4-tri-O-acetyl-β-D-glucopyranosid] uronate, was also tested. Morphine was used as a positive control to compare antinociceptive potencies of these compounds. Results. All compounds tested produced significant analgesic responses, as assessed by the tail flick model. Additionally, codeine 6-glucuronide showed significantly less immunosuppressive effects than codeine in vitro. Conclusions. We conclude that codeine 6-glucuronide and related compounds may have clinical benefit in the treatment of pain in immune compromised patients.

The effects of acute and repeated pyridostigmine bromide administration on response acquisition with immediate and delayed reinforcement

This experiment was designed to assess the effects of acute and repeated administration of pyridostigmine bromide (a carbamate with prophylactic and therapeutic uses) on response acquisition. Experimentally naïve, male Sprague-Dawley rats were exposed to a situation in which lever presses were either immediately followed by food-pellet presentation or after a 16-s resetting delay. Different groups of rats received either one acute administration of pyridostigmine bromide (10 mg/kg, by gavage) or repeated pyridostigmine administration for 7 days (1.5 mg/kg/day, by gavage). Other groups were treated with distilled water for the same period of time. Both acute and repeated pyridostigmine bromide administration decreased serum cholinesterase levels by approximately 50%, but neither treatment affected brain cholinesterase levels in our assay. Acute and repeated drug administration produced the same behavioral effects. Subjects exposed to the 0-s delay conditions obtained many more food pellets than those exposed to the 16-s delay conditions. Administration of pyridostigmine bromide delayed the onset of responding in some, but not all, of the subjects in the treated groups, independent of the delay condition to which they were exposed. Many more responses were observed on an inoperative lever during the 16-s delay conditions than during the 0-s delay conditions, especially during the 16-s delay condition in which subjects had received acute vehicle administration. Whether or not these effects of small doses of pyridostigmine bromide on response acquisition are of central or peripheral origin will need to be determined in future studies, as response acquisition in the present experiment may have been affected by pyridostigmines effects on gastrointestinal functioning and/or motor activity.

In vitro-in vivo myotoxicity of intramuscular liposomal formulations

AbstractPurpose. The first objective was to study the in vitro myotoxicity of empty liposomes and to examine whether liposome size, charge and fluidity affect liposome myotoxicity. The second objective was to investigate the effect of liposomal encapsulation on the in vitro and in vivo myotoxicity of loxapine compared to the loxapine commercial preparation (Loxitane®). Methods. The in vitro myotoxicity of empty liposomes and loxapine liposomes was evaluated by the cumulative efflux of the cytosolic enzyme creatine kinase (CK) from the isolated rat extensor digitorum longus (EDL) muscle over a 2 hour period. In the in vivo studies, the area under plasma CK curve over 12 hours was used to evaluate muscle damage. Results. The in vitro myotoxicity for all empty liposomal formulations was not statistically different from negative controls (untreated control muscles and normal saline injected muscles). However, these empty liposomal formulations were significantly less myotoxic than the positive controls (muscles injected with phenytoin and muscle sliced in half). In vitro-in vivo studies showed that the liposomal encapsulation of loxapine resulted in significant (P < 0.05) reduction in myotoxicity (80% in vitro and 60% in vivo) compared to the commercially available formulation which contains propylene glycol (70% V/V) and polysor-bate 80 (5% W/V) prepared at equal concentration. Conclusions. Results indicate that empty liposomes do not induce myotoxicity. Furthermore, liposomal size, charge and fluidity do not affect myotoxicity. In addition, in vitro and in vivo studies have demonstrated that liposomal encapsulation of loxapine can reduce myotoxicity compared to a formulation containing organic cosolvents.

Antisense inhibition of striatal GABAA receptor proteins decreases GABA-stimulated chloride uptake and increases cocaine sensitivity in rats

The functional status of striatal GABAA receptors appears to be inversely related to the magnitude of cocaine-induced behaviors. Exposure of striatum to antisense oligodeoxynucleotides (ASODNs) targeted to the mRNAs for the alpha 2 and the beta 3 subunits of the GABAA receptor should decrease expression of receptor proteins and therefore might be expected to increase cocaine sensitivity. ASODNs, scrambled ODNs or saline were injected into right lateral ventricle of rats and behavioral responses to cocaine were tested 18-20 h after treatment. Animals injected separately with alpha 2 or beta 3 ASODNs exhibited increased behavioral sensitivity to cocaine compared to rats injected with saline or scrambled ODNs including performing more 360 degrees turns to the left than to the right. There was significantly less GABA-stimulated Cl uptake in right striatum compared to left striatum of ASODN-treated rats with no significant difference between sides in control animals. Specific binding to benzodiazepine and convulsant sites on the GABAA receptor was not selectively altered by ASODN treatment. Combined alpha 2 beta 3 ASODN treatment did not affect either cocaine sensitivity or GABAA receptor function. There was no difference between the density of Nissl stained cells in the left and right edges of striatum in control or ASODN-treated rats indicating the absence of significant neurotoxic effects of the ASODN treatment. Injection of fluorescein-conjugated ASODNs indicated that ASODN is present in striatum at times during which behavioral and neurochemical indices of GABA receptor function are decreased. Thus, the functional status of GABAA receptors in striatum may be involved in determining cocaine sensitivity.

The Effects of Pyridostigmine Bromide and Permethrin, Alone or in Combination, on Response Acquisition in Male and Female Rats

It has been hypothesized that concurrent exposure to pyridostigmine bromide and permethrin may have contributed to the development of neurocognitive symptoms in Gulf War veterans. The present experiment was designed to investigate the effects of pyridostigmine bromide and permethrin alone, or in combination, on the acquisition of a novel response, one measure of normal cognitive functioning. Male and female Sprague-Dawley rats were treated with pyridostigmine bromide (1.5 mg/kg/day, by gavage in a volume of 5 ml/kg) or its vehicle for 7 consecutive days. They then also received an intraperitoneal injection of permethrin (0, 15, or 60 mg/kg) before they were exposed to an experimental session during which they could earn food by pressing a lever in an operant chamber. Serum permethrin levels increased as a function of its dose, and were higher in rats treated with pyridostigmine bromide. Sex differences were observed as permethrin levels were higher in female rats than in male rats following the highest dose. Pyridostigmine bromide delayed response acquisition in male and female rats, and resulted in higher response rates on the inactive lever in female rats than in male rats. Although permethrin levels were higher in subjects treated with pyridostigmine bromide than in those treated with vehicle, there were no differences in the behavioral effects of permethrin. Whether or not these behavioral effects of pyridostigmine bromide are of central or peripheral origin will need to be determined in future studies, as its effects on motor activity and/or gastro-intestinal motility may have affected response acquisition.