Comparative plasma disposition kinetics of albendazole and its new benzimidazol prodrug in dog.

The comparative pharmacokinetic behavior of albendazole (ABZ) and its new benzimidazol prodrug [1-tert-butyloxycarbonyl-5-propylthio-1-H-benzimidazol-2ylcarbamate of methyl] (ABZBoc), following their oral administration (10mg/kg) to healthy dogs was explored. Blood samples were obtained serially over a 24h period after treatment, then the plasma was analyzed by high-performance liquid chromatography (HPLC) to search the albendazole metabolites (ABZSO and ABZSO2). However, the albendazole parent drug was not detectable at any time after both treatments (ABZ and ABZBoc). By albendazole metabolites (ABZSO and ABZSO2) were the analytes recovered in the plasma after oral administration of ABZ and ABZBoc. Furthermore, some amounts of ABZBoc were also available in the plasma samples treated with this new produg. The plasma profile of each analyte followed a similar pattern after both treatments, the active metabolite (ABZSO) was the major analyte recovered in plasma (between 1 and 24h post-treatment). The pharmacokinetic parameters of both groups were calculated (Cmax, Tmax, t1/2, AUC0->∞), and analyzed using the Student's t-test, P<0.05. Thus,the pharmacokinetic analysis indicated four statistically significant changes in the pharmacokinetic parameters defined above of the albendazole metabolites (ABZSO, ABZSO2) between the group treated with albendazole (group A) and that treated with ABZBoc prodrug (group B). Hence, the levels of the various pharmacokinetics parameters were low in the group treated with prodrug, as well they did not reach equivalent concentrations to that of albendazole. These differences between albendazole and its new prodrug may be explained by the fact that ABZBoc prodrug was not effectively reduced in the intestine of dogs.

Relation between Serological Findings and Expression of IL-1ß and IL-6 genes in Patients Infected with Mycobacterium Tuberculosis in Iraq.

Mycobacterium Tuberculosis (TB) is one of the serious bacterial infections that cause diseases and may lead to death. In this study, 178 individuals were examined for TB infection at Baghdad TB center during the period from 15th January to 1st October 2021. Out of 178 participants, 73 were shown to be positive for TB infection, while 105 showed negative results. According to the results, there was no significant variation between infected males and females with TB in comparison to the control group (P>0.05). The results showed that the mean age of the patients for both males and females was in the range of 2-65 years. Additionally, there were significant differences in patients with TB compared to the control group in terms of the weight loss of 8.82 ± 6.75 Kg, red blood cell (RBC) count (3.43 ± 0.56) × 106/µl, white blood cell (WBC) count (3.12 ± 1.57) × 106/µl, platelet count (1.03 ± 0.56) × 106/µl, and hemoglobin level (6.66 ± 1.34) g/dl. A total of 30 TB patients and 50 normal individuals were genotyped to detect the IL-1ß rs 114534 gene. The polymerase chain reaction (PCR) was used for exon amplification in region 5 of the ILB1 gene in the TB patients by using specific primers. The finding showed that there was an amplified product of 249bp located in chromosome 2q13-14. A total of 30 TB patients and 50 normal individuals were also genotyped to detect the IL-6 rs 1800795 gene. The PCR was used for amplification of the IL-6 gene in TB patients by using specific primers. The finding showed that there was an amplified product of 431 bp located in chromosome 7p15-p2. The expression of the ILB1 gene was investigated in TB patients and healthy controls by using qPT-PCR. Results showed that there was a high Ct value for patients and controls with a high Ct value of templates, preoperational to the total ribonucleic acid (RNA) concentration and gene expression. The expression of the IL-6 gene was investigated in TB patients and healthy controls by using qPT-PCR. Our findings revealed a high Ct value for patients and controls with a high Ct value of templates, preoperational to the total RNA concentration and gene expression.

Impaired peripheral endothelial microvascular responsiveness in Alzheimer's disease.

BACKGROUND: The concept that the neurotoxicity of amyloid beta protein could partly result from vascular effects that may be detected in peripheral microcirculation is new. METHODS: We compared peripheral endothelial vascular responses of patients with early clinically confirmed Alzheimer's disease (AD) to that of people with normal cognition and those with other forms of dementia. Acetylcholine (ACh) was iontophoresed into the skin and the resultant vasodilator response was measured using laser Doppler flowmetery. RESULTS: The ratio of ACh response to saline (ratio E/S) was determined. Mean +/- SEM of ratios E/S were 8.8 +/- 0.9 for controls (n=168), 1.4 +/- 0.1 for AD patients (n=80) and 3.1 +/- 0.5 for other dementia (n=84). Using the optimal cut-off point of E/S ratio of 1.9, an 80% diagnostic sensitivity and specificity for AD have been observed. When the control sample was filtered for those with cardiovascular diseases and with MMSE < 28, this improved the specificity to 90% (n=119). Furthermore, 15 subjects were randomly drawn from a longitudinal healthy ageing study. Five of those subjects met the criteria for mild cognitive impairment (MCI) after eight years of follow up using a battery of cognitive tests. When tested for their E/S ratio in a blind fashion, the skin test successfully identified those subjects. CONCLUSIONS: The results support our hypothesis that endothelial alterations can be detected early in the course of the disease. We suggest that this simple skin test could potentially be applied as diagnostic adjunct in patients with mild cognitive symptoms or those with early clinical evidence of AD.

[Diagnostic modalities of visceral metastases during follow-up of patients with stage I-II melanoma]. / Malades suivis pour mélanome de stade I-II: modalités diagnostiques des métastases viscérales.

INTRODUCTION: Although not recommended in France at the consensus conference of 1994, routine monitoring of patients with stage I melanoma using imaging techniques is commonly carried out. The aim of this retrospective regional study was to define methods for diagnosing transition to the metastatic stage of melanoma. PATIENTS AND METHODS: This was a retrospective study based on questionnaires among dermatologists in the Champagne-Ardenne and southern Aisne regions of France. For each patient with stage IV melanoma between 1987 and 2002, data were collected concerning the primary melanoma (date of diagnosis, clinical picture, histopathologic features), stage of melanoma prior to diagnosis of metastatic melanoma and characteristics of the metastases (date, number, type, site and modern discovery: clinical signs or routine imaging). RESULTS: One hundred and eight patients (63 men and 45 women; mean age: 59 years) were included in the study. The predominant site of the primary melanoma was the trunk for men (n=31) and the lower limbs for women (n=16) and the mean Breslow index was 4.31 mm (SD=4.22), with histologic ulceration being present in 40% of cases. The mean time to transition to stage IV after discovery of the primary tumour was 2.8 years (SD=2.95). The modes of discovery of metastases comprised clinical examination (functional signs or physical examination) in 58 cases and routine imaging in 50 cases, with no significant differences based on whether patients were initially in stage I-II or in stage III. DISCUSSION: This study shows that over half of patients progressing to stage IV melanoma had a suspicious sign or clinical symptom, once again highlighting the importance of clinical monitoring. In contrast, many organ metastases, particularly pulmonary, were discovered by routine imaging examinations carried out as part of patient follow-up, although this is not currently recommended practice in France. CONCLUSION: The role of powerful imaging examinations such as scans, with constantly improving resolution, still remains to be defined in the follow-up of patients with stage I-II melanoma, and further prospective studies are thus required.

Influence of Genotype and Haplotype of MDR1 (C3435T, G2677A/T, C1236T) on the Incidence of Breast Cancer--a Case-Control Study in Jordan.

BACKGROUND: Breast cancer is the leading cause of cancer death among women and the second in humans worldwide. Many published studies have suggested an association between MDR1 polymorphisms and breast cancer risk. Our aim was to study the association between genetic polymorphism of MDR1 at three sites (C3435T, G2677A/T, and C1236T) and their haplotype and the risk of breast cancer in Jordanian females. MATERIALS AND METHODS: A case-control study involving 150 breast cancer cases and 150 controls was conducted. Controls were age-matched to cases. The polymerase chain reaction/restriction fragment length polymorphism (PCR- RFLP) technique and sequencing were performed to analyse genotypes. RESULTS: The distribution of MDR1 C3435T genotypes differed between cases and controls [cases, CC 45.3%, CT 41.3%, and TT 13.3%; controls, CC 13.4%, CT 43.3%, and TT 30.2%, p < 0.001]. Similarly, the distribution of G2677A/T significantly differed [cases, GG 43.1 %, GT+GA 50.9% and AA+TT 6%; controls, GG 29.6 %, GT+GA 50.9%, and AA+TT 19.4%, p = 0.004]. On the other hand, genotype and allelotype distribution of C1236T was not statistically different between cases and controls (p=0.56 and 0.26, respectively). The CGC haplotype increased the risk to breast cancer by 2.5-fold compared to others, while TGC and TTC haplotypes carried 2.5- and 5-fold lower risk of breast cancer, respectively. CONCLUSIONS: Genetic polymorphisms of MDR1 C3435T and G2677A/T, but not C1236T, are associated with increased risk of breast cancer. In addition, CGC, TGC and TTC haplotypes have different impacts on the risk of breast cancer. Future, larger studies are needed to validate these findings.

cis-Urocanic acid stimulates neuropeptide release from peripheral sensory nerves.

Previous studies using an antibody to cis-urocanic acid and mast-cell-depleted mice implicated both cis-urocanic acid and mast cells in the mechanisms by which ultraviolet B light suppresses systemic contact hypersensitivity responses in mice. In the absence of a direct stimulatory effect of cis-urocanic acid on connective tissue mast cells, an indirect association was investigated. A blister induced in the rat hind footpad was used to examine the effects of slowly perfused cis-urocanic acid on cutaneous blood flow. cis-Urocanic acid but not trans-urocanic acid increased microvascular flow by a mechanism largely dependent on the combined activity of the neuropeptides, substance P and calcitonin gene-related peptide. Perfusion of cis-urocanic acid over the base of blisters induced in sensory-neuropeptide-depleted rats did not have any stimulatory effect above that seen with perfusion of cis-urocanic acid together with neuropeptide receptor antagonists in control rats. There was a small direct effect of cis-urocanic acid on microvascular blood flow. As both substance P and calcitonin gene-related peptide could directly degranulate connective tissue mast cells, this study suggests that cis-urocanic acid indirectly activates mast cells via its effects on peripheral terminals of unmyelinated primary afferent sensory nerves. cis-Urocanic-acid-induced neuropeptides may also contribute to ultraviolet-B-induced cutaneous inflammation and alterations to Langerhans cell activity.

Elevation in plasma catecholamines in response to insulin stress is under both neuronal and nonneuronal control.

We have examined the contribution of neurogenic and nonneurogenic influences to the secretion of adrenal catecholamines (CA) in adult rats after iv administration of insulin. Plasma CA levels were measured in control rats and in rats after surgical or pharmacological adrenal denervation or adrenalectomy. Insulin-induced CA secretion was biphasic and proportional to the insulin dose used. The first phase was neurogenic in origin and produced a moderate increase in plasma adrenaline (A) levels, with little or no change in noradrenaline (NA) levels. This neurogenic increase in plasma A was reduced by partial denervation and abolished by surgical complete adrenal denervation, adrenalectomy, or administration of hexamethonium and atropine. The second phase occurred later and produced a dramatic increase in both plasma A and NA. This phase was initiated when the plasma glucose level fell below 75 mg/100 ml. This late release of A and NA was not altered by surgical or pharmacological adrenal denervation, showing that it was nonneuronal in origin. However, both the early and late phases were abolished by adrenalectomy, showing that adrenal secretion of CA was the origin of the increased plasma levels of NA and A. The late rise in plasma CA was also abolished by iv administration of glucose. These data suggest that the mechanism responsible for the nonneurogenic secretion of adrenal CA in response to insulin stress was sensitive to the level of hypoglycemia.

Mitochondrial DNA deletions parallel age-linked decline in rat sensory nerve function.

In rats, the function of sensory nerves in the hind limb declines significantly with age. Normally aging rats and rats treated neonatally with capsaicin were studied here. Quantification of vascular response and substance P in young (3 months) and old (24 months) rats showed additive effects of age and capsaicin treatment. The levels in dorsal root ganglion of a particular deletion in mitochondrial DNA (mtDNA(4834)) were about 300-fold higher in old compared to young rats. Capsaicin treatment had no significant effect on mtDNA(4834) abundance. Dorsal root ganglia of old (but not young) rats were found to contain a spectrum of multiple deletions. The abundance of mtDNA(4834) in dorsal root ganglia from individual rats correlated strongly with their decline in vascular function, even where vascular responses were systematically depressed due to prior capsaicin treatment. One possibility is that mitochondrial DNA mutations directly lead to functional decline at mitochondrial and tissue levels. Alternatively, loss of mitochondrial DNA integrity and physiological decline may be consequences of the same factor, such as oxidative stress.

Modulation of inflammation by reactive oxygen species: implications for aging and tissue repair.

Following tissue injury, adequate inflammatory vascular responses are essential for subsequent tissue repair. The aims of this study were to investigate the role of reactive oxygen species (ROS, generated at the injury site) in modulating the inflammatory response under acute- and chronic-injury conditions. The effect of age and the implications of this modulation for tissue repair was investigated. Using laser Doppler flowmetry, inflammatory vascular responses were monitored in the base of vacuum-induced blisters in the hind footpad of anesthetized rats (65 mg/kg Nembutal). Inflammation was amplified by superfusion of substance P (SP) over the blister base. The inflammatory response was examined in acute blisters induced on either naïve skin (acute-injury model) or on skin innervated by a chronically injured nerve (chronic-injury model). Furthermore, the acute-injury model was examined during early and late phases, 0 and 5 h after blister induction, respectively. The involvement of ROS was assessed by either combined superfusion of the antioxidants: superoxide dismutase and catalase over the blister base in acute-injury, or intramuscular injection of tirilazad in chronically injured rats. The results showed that antioxidant treatment had no effect on the response during early and late phases of acute inflammation in young rats. However in old rats, the vascular response was significantly attenuated (60%) or significantly increased (40%) during the early and late phases of acute inflammation, respectively. Under chronic-injury conditions, antioxidant treatment significantly enhanced the response in both young and old rats. We then examined the effect of antioxidant, tirilazad, on the healing of a full thickness thermal injury induced in the intrascapular region (using a CO(2) laser) of the rat. Following burn injury, tirilazad was injected around the wound site starting on day 1 (early treatment) or day 6 (late treatment). Tirilazad had opposing actions on wound closure with early and late treatments delaying (24.6 +/- 0.6 d) or accelerating (14.2 +/- 0.3 d) wound closure compared with the group of aged controls (20.3 +/- 0.8 d). The results suggest that ROS have a paradoxical role exerting either a positive or negative effect on the inflammatory response with age. We contend that the role of ROS in modulating inflammation should be considered when designing treatment protocols to accelerate tissue repair.

A role for free radicals and nitric oxide in delayed recovery in aged rats with chronic constriction nerve injury.

Using a reversible chronic constriction injury (CCI) model of neuropathic pain, we previously demonstrated that changes in thermal hyperalgesia correlate with the changes in peripheral microvascular blood flow in the affected paw, and that recovery can be assessed by normalization of both behavioral and vascular responses. Using the same model, this study examined age-related changes in recovery after nerve injury and the involvement of free radicals and nitric oxide (NO) in these changes. Four loose, nonconstrictive ligatures were applied to the sciatic nerve in the right, mid-thigh region of young and old (3 and 24 months) Sprague Dawley rats. All rats were monitored weekly (for 8-10 weeks) for their thermal threshold using a 46 degrees C water bath and some groups were used to examine endothelial and smooth muscle-dependent microvascular responses to substance P (SP) and sodium nitroprusside (SNP), respectively. These substances were perfused over the base of blisters raised on the footpad innervated by the injured nerve. Free radical activity in the sciatic nerve was assessed by measuring the activity of xanthine oxidase (XO) and lipid hydroperoxides (LPO). Young rats showed signs of recovery (reduction in thermal hyperalgesia and improvement of peripheral microvascular blood flow) from the fifth week. No signs of recovery were observed in old rats for 8 weeks, with some reduction in thermal hyperalgesia observed by weeks 9 and 10. XO activity was significantly higher in young injured nerves compared to sham (400%) and was even significantly greater in old injured nerves (680%). Similarly, old injured nerves showed 300% increase in LPO levels compared to sham. The role of reactive oxygen species (ROS) in delayed recovery in old rats was examined using the antioxidant tirilazad mesylate. Tirilazad (20 mg/kg) was injected intramuscularly (im) in the mid-thigh region starting on day 1 post CCI, (early treatment) or day 7 (late treatment). Levels of LPO in the injured sciatic nerves were significantly reduced using either early or late treatment, however tirilazad had opposing effects on recovery, prolonging or alleviating thermal hyperalgesia, respectively. The role of neuronal nitric oxide (nNO) was then examined using the specific neuronal nitric oxide synthase (nNOS) inhibitor, 3-bromo-7-nitroindazole (3Br-7NI) (10 mg/kg). 3Br-7NI resulted in a significant alleviation of thermal hyperalgesia with improvement in the vascular responses from weeks 5 and 6 onwards. A combination of 3Br-7NI and tirilazad treatment was also used but did not show an additive effect. The results suggest that ROS and nNO contribute to delayed recovery of injured nerves in old rats and to the maintenance of thermal hyperalgesia and the reduction in microvascular blood flow in the area innervated by the injured nerve. The results also raise the notion that possible interaction of free radicals with NO to form peroxynitrite might be responsible for such delayed recovery. Ironically, this study also reveals a positive role for free radicals in tissue repair and raises the notion that early intervention with antioxidants could exert a negative effect on repair of injured nerves.

Age-related differences in the time course of capsaicin-induced hyperalgesia.

The effect of age on hyperalgesia, one of the most common signs of injury, has not been previously examined in humans. A psychophysical study was conducted in 10 young (26.9+/-4.6 years) and 10 older (79. 0+/-5.7 years) healthy volunteers to investigate the effect of age on the development of hyperalgesia induced by topical application of capsaicin (0.1 ml, 5 mg/ml). The capsaicin patch (diameter 2 cm) was applied for 1 h. The intensity of capsaicin-induced spontaneous sensation, mechanical pain threshold, area of flare, heat and punctate hyperalgesia were measured hourly for 3 h after the application. Older adults took a longer period to report first pain. There was no age effect on the magnitude of spontaneous sensation, flare size and area of heat hyperalgesia. The area of heat hyperalgesia rapidly decreased over time in both age groups. In marked contrast, the area of punctate hyperalgesia and associated reduction in the mechanical pain threshold were maintained in older adults over the entire 3 h test period, but resolved rapidly in young adults. We conclude that, given the same intensity of noxious stimulation, older adults display a similar magnitude of hyperalgesia as younger persons. However, once initiated, punctate hyperalgesia appears to resolve more slowly in older people. This finding may indicate age differences in the plasticity of spinal cord neurons following an acute injury.

Hashish: synthesis and central nervous system activity of some novel analogues of cannabidiol and oxepin derivatives of delta 9-tetrahydrocannabinol.

Several C-10 substituted cannabidiol (CBD) derivatives and novel oxepin derivatives of delta 9-tetrahydrocannabinol (delta 9-THC) were synthesized and evaluated for biological activity in mice and dogs. Treatment of 10-bromocannabidiol diacetate (3) with various amines in Me2SO gave the corresponding 10-aminocannabidiol derivatives 4-6. Similarly, treatment of 3 with NaN3 gave the azido compound 7, which with LiA1H4 afforded the 10-aminocannabidiol 9. However, reduction of 7 with CrCl2 formed the amide 8, which on further reduction with LiA1H4 gave the N-ethyl analogue 10. Coupling of 9 with 11 in the presence of dicyclohexylcarbodiimide formed 12, which was then deprotected with HCl to give the analogue 13. The oxepin analogue 14a was synthesized from 3 by treatment with Na2CO3 in CH3OH/H2O at room temperature. The dimethylheptyl analogue 14b was similarly prepared. Incorporation of N-ethyl (10), N-methyl-N-propargyl (6), and morpholino (4) groups in CBD at position 10 resulted in analogues that were more potent than CBD in producing hypoactivity in mice. These analogues had relatively little effect on rectal temperature. Selected substitutions at C-10 also resulted in analogues that were partially effective in blocking delta 9-THC antinociceptive activity. This blockade was observed particularly in compound 10, which also showed unusually toxic properties. Incorporation of a seven-membered oxepin in the delta 9-THC structure eliminated cannabinoid activity although substitution of the pentyl side chain with a 1,2-dimethylheptyl in the oxepin 14b resulted in CNS depression in mice.

CGRP and nitric oxide of neuronal origin and their involvement in neurogenic vasodilatation in rat skin microvasculature.

1. Sensory nerves are important for the initiation of neurogenic inflammation and tissue repair. Both calcitonin gene-related peptide (CGRP) and nitric oxide (NO) have been implicated in neurogenic vasodilatation and inflammatory responses. 2. A blister model in the rat hind footpad was used as a site to induce neurogenic vasodilatation in response to antidromic electrical stimulation of the sciatic nerve. Blood flux was monitored with a laser Doppler flow monitor. 3. The quantitative contributions of CGRP and NO to vasodilatation were examined by use of the CGRP receptor antagonist CGRP8-37 and NO synthase inhibitors 7-nitroindazole (7-NI), 3-bromo 7-NI and N(G)-nitro L-arginine methyl ester (L-NAME). The potential modulatory role of endothelin was examined by use of the ET(A) receptor antagonist BQ-123. 4. CGRP8-37 (10 microM) was perfused over the blister base before nerve stimulation and continuously throughout the post-stimulation period, resulting in a significant reduction (41%) in the blood flux vascular response. 5. Pretreatment with the specific neuronal NO synthase inhibitors, 7-NI and 3-bromo 7-NI (10 mg kg(-1), i.v.), and of the non-specific L-NAME (100 microM), resulted in significant inhibition of the blood flux response (36%, 72% and 57% decrease, respectively). In contrast, 7-NI treatment in young rats pretreated with capsaicin had no further effect on the vascular response, suggesting that the source of NO is the sensory nerves. 6. BQ-123 (10 microM) significantly enhanced the stimulation-induced blood flux response (61% increase). When 7-NI was co-administered with either CGRP8-37 or BQ-123, the drug actions were additive, suggesting that there was no interaction between NO and CGRP or endothelin. 7. These data suggest that both NO and CGRP participate in neurogenic vasodilatation in rat skin microvasculature and that this response is modulated by endogenous endothelin.

Nitric oxide and sensory nerves are involved in the vasodilator response to acetylcholine but not calcitonin gene-related peptide in rat skin microvasculature.

1. The contributions of sensory nerves and nitric oxide (NO) to vasodilator responses to acetylcholine (ACh) and calcitonin gene-related peptide (CGRP) were examined in rat skin microvasculature with a laser Doppler flowmeter to monitor relative blood flow. 2. Perfusion of ACh (100 microM; for 30 min) over a blister base on the rat hind footpad elicited microvascular vasodilatation and this response was not sustained. CGRP (1 microM; 10 min perfusion) also elicited vasodilatation and this response was maintained even when CGRP was no longer in contact with the blister base. 3. The vasodilator response to ACh was significantly smaller in rats pretreated as neonates with capsaicin to destroy primary sensory afferents than it was in age-matched controls. The vasodilator response to CGRP was unaffected by capsaicin pretreatment. 4. Selective inhibitors of NO synthase, NG-nitro-L-arginine (L-NOARG) and NG-monomethyl-L-arginine (L-NMMA) (both at 100 microM) attenuated the vasodilator response to ACh in control rats, but had no effect on the vasodilator response to CGRP. There was a significant L-NOARG-resistant component in control rats while in capsaicin-treated rats the vasodilator response to ACh was virtually abolished by L-NOARG. The inactive stereoisomer NG-monomethyl-D-arginine (100 microM) did not affect the vasodilator response to ACh. 5. The efficacy of L-NOARG and L-NMMA as inhibitors of endothelium-dependent responses was confirmed by use of an endothelium-dependent vasodilator, the calcium ionophore A23187 (100 microM; 10 min perfusion). Vasodilatation to A23187 was strongly attenuated by both L-NOARG and L-NMMA.6. These results suggest that sensory nerves and NO are both involved in the dilatation produced by ACh in rat skin microvasculature. A component of the vasodilator response elicited by ACh involves a direct action on the microvascular endothelium with subsequent generation of NO, while an additional component is elicited via activation of sensory nerves. The vasodilator mediator(s) released by ACh from sensory nerves acts largely independently of NO.7. The vasodilator response to CGRP is independent of a prejunctional action on sensory nerves and of NO.

Drugs from the sea: conotoxins as drug leads for neuropathic pain and other neurological conditions.

The oceans are a source of a large group of structurally unique natural products that are mainly found in invertebrates such as sponges, tunicates, bryozoans, and molluscs. It is interesting to note that the majority of marine compounds currently in clinical trials or under preclinical evaluation are produced by these species rather than as secondary metabolites by marine algae. Through the combined efforts of marine natural products chemists and pharmacologists a number of promising compounds have been identified that are either already at advanced stages of clinical trials such as the new anti-cancer drug marine alkaloid ecteinascidin 743, or have been selected as promising candidates for extended preclinical evaluation. This is the case for conotoxins, (Table 1) where a number of conopeptides are currently being developed as analgesics for the treatment of neuropathic pain.

Sensory peptides as neuromodulators of wound healing in aged rats.

An intact nociceptor system of primary afferent sensory nerves is important for the initiation of the inflammatory process and successful tissue repair. Dysfunction of this system with age could be a contributing factor for delayed wound healing in the elderly. This study was designed to examine the role of sensory nerves (using capsaicin-pretreated rats) and the effect of aging on the healing of a thermal wound induced on the interscapular region (using a CO2 laser). The healing endpoint was the time when full wound contraction had occurred. The ability of the sensory peptides, substance P (SP) and calcitonin gene-related peptide (CGRP), in modulating wound healing in aged rats was examined by taking into account the modulatory interaction effects between these peptides. A blister model in the rat hind footpad combined with a laser Doppler flowmeter was used initially to establish these vascular interaction effects. The results showed a significant delay in thermal wound healing in both the capsaicin-treated and aged rats. Using the blister model, we demonstrated the ability of SP and CGRP to induce a limited and a prolonged vasodilator response, respectively. When tested together, SP attenuated the vascular effect of CGRP, an effect that was reversed using the tachykinin antagonist spantide II. When injected at the wound site, SP and CGRP were equally potent in promoting wound healing in aged rats. The beneficial effect of taking the interaction effects into account was most evident in a group of rats that received the tachykinin antagonist as part of their initial treatment with CGRP and 6 h later, an injection of SP; the time to complete wound closure was 11.3 +/- 0.6 days compared to 21.0 +/- 0.9 days in aged controls. We contend that would healing in aged rats can be accelerated by exogenous administration of sensory peptides. Furthermore, modulatory interaction effects between sensory peptides should be taken into consideration when designing any treatment protocol that purports to accelerate wound healing.

Effects of aging on neurogenic vasodilator responses evoked by transcutaneous electrical nerve stimulation: relevance to wound healing.

We have previously shown an age-related decline in the modulation of skin vascular reactivity by sensory nerves that correlates with a decline in wound repair efficacy. This study was designed to examine the possibility that improving the functional ability of aged sensory nerves using noninvasive transcutaneous electrical nerve stimulation (TENS) could also accelerate tissue repair. TENS of the sciatic nerve, combined with measuring blood flow responses in the rat hind-footpad using laser Doppler flowmetry, was used to establish the vascular effects. Following TENS (using parameters 20V, 5 Hz for 1 min), similar increases in vascular responses were obtained in both young (13.2+/-0.9 cm2) and old rats (11.6+/-2.3 cm2). In contrast, capsaicin-pretreated rats showed markedly diminished responses. Sympathetic fibers did not appear to modulate these sensory nerve responses. In the second part, a thermal wound was induced (using a CO2 laser) in the interscapular region of old rats (under anesthesia). In the active treatment group, TENS was applied twice daily for the initial 5 days, and the sham group received inactive TENS. Using the healing endpoint as the time when full wound contraction occurred, the active group required 14.7+/-0.2 days for complete healing, a significant improvement over the sham group (21.8+/-0.3 days). We contend that low-frequency TENS can improve the vascular response of old rats. In addition, wound healing in aged rats can be accelerated by peripheral activation of sensory nerves at low-frequency electrical stimulation parameters.

Differential involvement of conotoxin-sensitive mechanisms in neurogenic vasodilatation responses: effects of age.

During aging there is a decline in sensory nerve function that is associated with reduced neurogenic inflammation and poor wound repair. The cellular mechanism(s) responsible for this decline in function with age is not well understood. We previously reported that sensory nerves in aged rats release sensory neuropeptides preferentially in response to low-frequency (5 Hz) as compared with higher-frequency (15 Hz) antidromic electrical stimulation, and that low-frequency transcutaneous electrical nerve stimulation accelerates wound healing. The present study investigates possible mechanisms for this preferential response. Using laser Doppler techniques, we have measured changes in blood flow in the base of vacuum-induced blisters induced in the rat hind footpad of young and old animals in response to low-frequency (5 Hz) or high-frequency (15 Hz) electrical stimulation (20 V, 2 ms for 1 minute) of the sciatic nerve. The relative contributions of the sensory neuropeptides, substance P and calcitonin gene-related peptide (CGRP), and of N-type voltage-gated calcium channels to the vascular responses were assessed by using the specific receptor antagonists RP67580, which is 2-(1-imino-2-(2 methoxy phyenyl) ethyl)-7,7 diphenyl-4 perhydroisoindolone-(3aR, 7aR); CGRP(8-37); and omega-conotoxin GVIA (Conus geographus), respectively. The results showed a greater involvement of substance P at high-frequency electrical stimulation and of CGRP at low-frequency stimulation. Our finding that omega-conotoxin-sensitive N-type calcium channel function was preserved with age and was only involved in the vascular response to low-frequency electrical stimulation could explain our previous report demonstrating beneficial effects of low-frequency transcutaneous electrical nerve stimulation to wound repair in aged animals. The current results have important practical implications for improving tissue repair in the aged.

Modulation of a peripheral inflammatory response to substance P by locally administered opioid receptor agonists.

Using a blister model of inflammation in the rat hind footpad, the present study was undertaken to examine possible peripheral effects of specific mu (DAGO) and delta (DSLET) opioid receptor agonists on an inflammatory response induced by substance P, the putative mediator of neurogenic inflammation. When perfused over the blister base, SP induced both plasma extravasation and vasodilatation responses. These responses were significantly inhibited in the presence of either opioid receptor agonist in a naloxone reversible manner. DSLET inhibited SP responses in a dose dependent manner and was 100 times more potent than DAGO. The role of primary afferent sensory nerve terminals in these modulatory effects was investigated in rats pretreated as neonates with capsaicin. The ability of DAGO and DSLET to inhibit the inflammatory response in these rats was significantly less than that in controls. The data raises the possibility that the inhibitory effect of the opioid receptor agonists on the inflammatory response might reflect a role for opioids in modulating tachyphylaxis to SP.

Substance P induced hydrolysis of inositol phospholipids in rat skin in an in vivo model of inflammation.

The present study was undertaken to study the ability of substance P (SP) to induce inositol phospholipid (IP) hydrolysis measured as inositol mono-phosphate (IP1) accumulation, in an in vivo blister model of neurogenic inflammation in the rat hind footpad. SP was found to induce IP1 accumulation in a concentration dependent manner. The use of SP analogues (SP5-11 and SP1-7) indicated that the response is mainly mediated by the C-terminal sequence of the peptide. The response was significantly reduced by the SP antagonist spantide, suggesting that the response is mostly due to activation of the SP receptor on small diameter vessels. Capsaicin pretreatment did not have an effect on the ability of SP to induce the response. Experiments with mepyramine suggest that the response is also partly mediated by SP induced histamine release from mast cells. This is the first study to provide direct evidence for phosphoinositide mediated SP effects in the skin.