New Delivery Route of Gambogic Acid Via Skin for Topical Targeted Therapy of Cutaneous Melanoma and Reduction of Systemic Toxicity

Cutaneous melanoma is the deadliest form of skin cancer, and gambogic acid (GA) exhibits potent anti-melanoma activity. However, clinical application of GA via intravenous injection and oral administration is limited by systemic toxicity and rapid metabolism in the blood. Here, we developed a new, topical route of GA delivery for anti-melanoma activity and reduction of systemic toxicity. The results indicated that the barrier of the stratum corneum (SC) and low diffusion of GA in the hydrophilic viable skin (epidermis and dermis) limited the GA penetration through intact skin. The combination of azone (AZ) and propylene glycol (PG) showed obvious synergistic effects on skin penetration by GA via improving the permeability of the SC and greatly increasing the skin accumulation of GA, thereby forming a high drug concentration in the skin and achieving a topical targeted treatment of melanoma. In addition, GA (AZ–PG) achieved the same anti-melanoma effect via topical delivery as via intravenous injection. Intravenous injection and oral administration of GA induced remarkable pathological changes in various organs in mice, whereas GA was not toxic to various organs or to the skin via topical delivery. These findings indicated that topical administration of GA is an alternative route for melanoma treatment.     

A lupus-like reaction to subcutaneous interferon-α at injection sites

Interferon-α has been associated with a wide range of adverse events (AEs). A lupus-like reaction at the injection site of subcutaneous (SC) interferon-α is exceptionally rare. A 60-year-old woman with recurrent metastatic melanoma repeatedly developed cutaneous lupus-like reactions at the SC interferon-α-2b injection sites on her thighs. Known features of lupus-like reactions at SC interferon-α injection sites are reviewed, and cutaneous injection site reactions to SC interferon-α are summarized.     

Nociceptin/orphanin FQ (N/OFQ) induces a quasi-morphine abstinence syndrome in the rat

Nociceptin/orphanin FQ (N/OFQ) is a neuropeptide that exerts antiopiate effects under some circumstances, and there is evidence that it contributes to opiate tolerance. This raises the question, might N/OFQ also contribute to opiate dependence and abstinence? Twenty-two male Sprague-Dawley rats were cannulated in the third ventricle and challenged 7 days later by third ventricle injection of 50, 200 or 1,000 ng N/OFQ or saline alone. Each rat was observed under "blind" conditions for 30 min beginning 15 min after onset of the third ventricle injection. There was a significant positive linear trend of signs as a function of N/OFQ dose. Subjects receiving saline had 18.0+/-2.0 (mean+/-SEM) overall abstinence-like signs, whereas subjects receiving 50, 200 or 1000 ng N/OFQ had 35.2+/-3.6, 49.8+/-2.6 and 63.5+/-9.7 signs, respectively. In 16 additional rats, abstinence-like signs induced by 1000 ng N/OFQ were significantly attenuated by low SC doses of morphine or clonidine. These results raise the possibility that N/OFQ might contribute to opiate dependence and subsequent abstinence syndrome. On the other hand, N/OFQ over a wide dose range induced abstinence signs with similar potency in morphine dependent and non-dependent rats.     

Skin targeted DNA vaccine delivery using electroporation in rabbits. I: efficacy

Genetic immunization through skin is highly desirable as skin has plenty of antigen presenting cells (APCs) and is easily accessible. The purpose of this study was to investigate the effects of electroporation pulse amplitude, pulse length and number of pulses on cutaneous plasmid DNA vaccine delivery and immune responses, following intradermal injection in vivo in rabbits. Expression of the delivered plasmid was studied using a reporter plasmid, coding for beta-galactosidase. The efficiency of DNA vaccine delivery was investigated using a DNA vaccine against Hepatitis B, coding for Hepatitis B surface antigen (HBsAg). Serum samples and peripheral blood mononuclear cells (PBMC) were analyzed for humoral and cellular immunity, respectively, following immunization. The expression of transgene in the skin was transient and reached its peak in 2 days post-delivery with 200 and 300 V pulses. The expression levels with 200 and 300 V pulses were 48- and 129-fold higher, respectively, compared with the passive on day 2. In situ histochemical staining of skin with X-gal demonstrated the localized expression of beta-galactosidase with electroporation pulses of 200 and 300 V. Electroporation mediated cutaneous DNA vaccine delivery significantly enhanced both humoral and cellular immune responses (p<0.05) to Hepatitis B compared to passive delivery. The present study demonstrates the enhanced DNA vaccine delivery to skin and immune responses by topical electroporation. Hence, electroporation mediated cutaneous DNA vaccine delivery could be developed as a potential alternative for DNA vaccine delivery.     

Doxorubicin skin penetration from monoolein-containing propylene glycol formulations

Topical chemotherapy with the antineoplastic doxorubicin (DXR) could be an alternative to treat skin cancer, however its poor skin penetration often limits the efficacy of topical formulations. The aim of this work was to study the effect of monoolein (MO), a penetration enhancer, on the in vitro skin permeation and retention of DXR. DXR was incorporated in a propylene glycol preparation containing 0-20% of MO. DXR release rate and topical delivery were evaluated in vitro using acetate cellulose membrane and porcine skin, respectively, mounted in a Franz diffusion cell. At 5%, MO did not significantly change DXR release rate, but MO concentrations larger than 10% decreased almost twice its release. In vitro skin penetration studies showed that the presence of MO in the propylene glycol formulations markedly increased DXR presence in the stratum corneum (SC). At 5%, MO significantly increased the amount of DXR in the SC already in the first hours, attained a maximum in 6h. Comparing propylene glycol formulations containing more than 10% MO with that containing 5%, the former took the double of the time (12h) to reach the same amount of DXR in the skin, result that is in agreement with in vitro release studies. Interesting, despite the fact that MO significantly increased the amount of DXR in the SC, drug transdermal delivery did not change. These findings suggest a cutaneous delivery of DXR that is an important condition for topical treatment of skin tumors. Further in vivo experiments can show DXR delivery to deeper skin layers.     

Acute intraperitoneal cholecalciferol (vitamin D3) toxicosis in mice: its nature and treatment with diverse substances

To study the overt toxicosis of intraperitoneally (IP)-administered single doses of cholecalciferol (D3), groups of male CF-1 mice (N = 12) were given graded doses of D3 in corn oil and observed for 21 days. There was a 2- to 4-day onset of signs, including ocular squinting, reluctance to move, lethargy, weakness, anorexia, hunched posture, rough haircoat, and dehydration. This was followed by tremors, coma, and death (large doses) or gradual recovery. Deaths occurred 3 days (larger doses) to 21 days after D3 injection. The linear regression of mortality probits on log10 dose was Y = 7.332X-10.653. The median lethal dose (LD50) of D3 and 95% confidence limits were 135.4 mg/kg (112.2-157.4 mg/kg). To screen potential antidotes against acute D3 toxicosis, groups of mice (N = 12) were given subcutaneous (SC) injections of various substances beginning 2 days after IP injection of a large dose of D3 (300 mg/kg). Substances were given once or twice daily in constant volumes of saline solution (66.8 ml/kg) for 7 days. Two control groups were given D3 but no treatment. They both had 91.7% mortality; their mean (+/- SD) survival time (MST: censored to 21 days observation) was 6.8 +/- 4.7 days and 10.3 +/- 7.0 days. Mortality and MST were not affected significantly (P greater than 0.05) by once-daily injection of saline solution, saline containing dexamethasone (DEX), or saline containing the following substances with or without DEX: ascorbate; citrate; dimercaptosuccinic acid; oxytetracycline; ZnSO4; or MgCl2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ultrasound and transdermal drug delivery

Transdermal drug delivery offers an attractive alternative to the conventional drug delivery methods of oral administration and injection. However, the stratum corneum acts as a barrier that limits the penetration of substances through the skin. Application of ultrasound to the skin increases its permeability (sonophoresis) and enables the delivery of various substances into and through the skin. This review presents the main findings in the field of sonophoresis, namely transdermal drug delivery and transdermal monitoring. Particular attention is paid to proposed enhancement mechanisms and future trends in the field of cutaneous vaccination and gene delivery.     

Pyrogenicity of polyadenylic · polyuridylic acid in rabbits

Summary Polyadenylic polyuridylic acid injected intraveneously into rabbits produced a rapid-onset, monophasic fever. Pyrogenic tolerance occurred in rabbits following daily injections of polyadenylic polyuridylic acid. However, direct injection of the agent into the preoptic anterior hypothalamic region of rabbit's brain produced a markedly different fever. After an intrahypothalamic injection of polyadenylic - polyuridylic acid, fever was delayed in onset and persisted for a longer period. At room temperature, the fever was due to both increased metabolism and cutaneous vasoconstriction. In a colder atmosphere the fever was due solely to increased metabolism, whereas in the heat the fever was due to reduction in cutaneous blood flow and respiratory evaporative heat loss. In addition, the fever induced by intravenous polyadenylic · polyuridylic acid injection was reversed by a cyclooxygenase inhibitor, but not by a protein synthesis inhibitor. Polyadenylic - polyuridylic acid was shown to stimulate PGE₂ production from rabbit's hypothalamus in vitro. The results reveal that this agent is a prostaglandin-dependent pyrogen.This study was supported by grants from the National Science Council of the Republic of China Send offprint requests to M.-T. Lin at the above address     

Intranasal administration of dry powder anthrax vaccine provides protection against lethal aerosol spore challenge

The use of an aerosolizable form of anthrax as a biological weapon is considered to be among the most serious bioterror threats. Intranasal (IN) delivery of a dry powder anthrax vaccine could provide an effective and non-invasive administration alternative to traditional intramuscular (IM) or subcutaneous (SC) injection. We evaluated a dry powder vaccine based on the recombinant Protective Antigen (rPA) of Bacillus anthracis for vaccination against anthrax via IN immunization in a rabbit model. rPA powders were formulated and administered IN using a prototype powder delivery device. We compared serum IgG and toxin neutralizing antibody (TNA) titers of rabbits immunized IN with 10 microg rPA of a powder formulation with those immunized with the same dose of liquid rPA vaccine, delivered either IN or by IM injection. In addition, each group was tested for survival after aerosol spore challenge. Our results showed that IN vaccination with rPA powders elicited serum PA-specific IgG and TNA titers that were equivalent to those raised by liquid rPA administered IN. Serum PA-specific IgG and TNA titers after IN delivery were lower than for IM injection, however, after aerosol spore challenge, rabbits immunized IN with powders displayed 100% protection versus 63% for the group immunized IN with the liquid vaccine and 86% for the group immunized by IM injection. The results suggest that an IN powder vaccine based on rPA is at least as protective as a liquid delivered by IM injection.     

Investigation of Drug Delivery in Rats via Subcutaneous Injection: Case Study of Pharmacokinetic Modeling of Suspension Formulations

With the rising cost of drug research, “do more with less” has become a new emphasis in the pharmaceutical industry. Consequently, the early analysis of pharmacokinetic/pharmacodynamic, efficacy, and safety parameters for a new drug target is critical for ensuring informed decision-making as soon as possible during the drug discovery process. When absorption, distribution, metabolism, and excretion properties of compounds are suboptimal which is especially true during the early stages of drug discovery, obtaining the desired exposure can be challenging via the most common routes (oral, intravenous). Therefore, subcutaneous (SC) injection is often explored as an alternate route of delivery. Although SC injection is used widely in the industry, information about how to model and predict the absorption of drugs administered via SC injection is not readily available. In the current research, we analyzed the absorption behavior of 12 model compounds covering a wide range of physicochemical properties following SC injection. We introduced a compound-specific parameter, the absorption factor from single SC injections of suspension doses of each compound, to aid in modeling and predicting of drug absorption profiles. The pharmacokinetic models derived in this study are capable of describing and predicting the absorption properties of SC injection for individual compounds.     

Controllable continuous sub-tenon drug delivery of dexamethasone disodium phosphate to ocular posterior segment in rabbit

Corticosteroids have been used for treatment of posterior segment eye diseases, but the delivery of drug to the posterior segments is still a problem to resolve. In our study, we explore the feasibility of Sub-tenon’s Controllable Continuous Drug Delivery to ocular posterior segment. Controllable continuous sub-tenon drug delivery (CCSDD) system, intravenous injections (IV) and sub-conjunctival injections (SC) were used to deliver dexamethasone disodium phosphate (DEXP) in rabbits, the dexamethasone concentration was measured in the ocular posterior segment tissue by Shimadzu LC-MS 2010 system at different time points in 24?h after first dose injection. Levels of dexamethasone were significantly higher at 12, 24?h in CCSDD than two other approaches, and at 3, 6?h in CCSDD than IV in vitreous body (p?p?p?0–24 in CCSDD group is higher than two other groups in all ocular posterior segment tissue. Our results demonstrated that dexamethasone concentration could be sustained moderately higher in the posterior segment by CCSDD than SC and IV, indicating that CCSDD might be a therapeutic alternative to treat a variety of intractable posterior segment diseases.     

Minimally invasive cutaneous delivery of macromolecules and plasmid DNA via microneedles

The stratum corneum (SC) represents a significant barrier to the delivery of gene therapy formulations. In order to realise the potential of therapeutic cutaneous gene transfer, delivery strategies are required to overcome this exclusion effect. This study investigates the ability of microfabricated silicon microneedle arrays to create micron-sized channels through the SC of ex vivo human skin and the resulting ability of the conduits to facilitate localised delivery of charged macromolecules and plasmid DNA (pDNA). Microscopic studies of microneedle-treated human epidermal membrane revealed the presence of microconduits (10-20 microm diameter). The delivery of a macromolecule, beta-galactosidase, and of a 'non-viral gene vector mimicking' charged fluorescent nanoparticle to the viable epidermis of microneedle-treated tissue was demonstrated using light and fluorescent microscopy. Track etched permeation profiles, generated using 'Franz-type' diffusion cell methodology and a model synthetic membrane showed that >50% of a colloidal particle suspension permeated through membrane pores in approximately 2 hours. On the basis of these results, it is probable that microneedle treatment of the skin surface would facilitate the cutaneous delivery of lipid:polycation:pDNA (LPD) gene vectors, and other related vectors, to the viable epidermis. Preliminary gene expression studies confirmed that naked pDNA can be expressed in excised human skin following microneedle disruption of the SC barrier. The presence of a limited number of microchannels, positive for gene expression, indicates that further studies to optimise the microneedle device morphology, its method of application and the pDNA formulation are warranted to facilitate more reproducible cutaneous gene delivery.     

Inhaled insulin

Inhalation of regular insulin for meal time glucose control has been found to be safe, efficacious and reliable in Type I and Type II diabetics. The administration of regular insulin through the human lungs by inhalation has been conducted in at least 14 short studies in both normal and diabetic subjects beginning as early as 1925. In all studies, significant insulin absorption and lowering of blood glucose was observed in the absence of penetration enhancers. Although a concern of variable dosing was raised in early studies, the development of new reproducible delivery systems has ensured that the variability of aerosol insulin can be as good, if not better, than subcutaneous (SC) injection. In the longest controlled studies in humans to date, both Type I and Type II insulin-dependent diabetics used a novel inhaled dry powder insulin delivery system for 3 months for meal time glucose control. The study results indicate that inhaled insulin provides equivalent glucose control, measured by hemoglobin A1c, when directly compared to SC injection. Interim results from an additional study with Type II diabetics who were failing oral hypoglycemic agents suggest that adjunctive therapy with inhaled insulin markedly improved glycemic control with a low risk of hypoglycemia. In all the 3 month studies the system was efficacious, well tolerated, well liked, and resulted in reproducible results. A potential advantage of aerosol insulin is that it is more rapidly absorbed (serum peak at 5-60 min) and cleared than SC injection (peak at 60-150 min), which provides a more relevant and convenient therapy for meal time glucose control. The relative efficiency of insulin delivery by aerosol, compared to SC injection, has been estimated from the dose measured at the exit point of the aerosol device, and found to range between 8 and 25% of SC, depending on the study.     

Bremazocine-induced backwards walking behavior in rats is mediated via opioid kappa receptors

Bremazocine dose-dependently induced backwards walking behavior in rats after its SC injection. Only the (-) but not the (+) enantiomer induced backwards walking. Pretreatment with either naloxone or MR 2266 reduced the bremazocine-induced backwards walking. MR 2266 was at least ten times more potent than naloxone. These findings suggest that bremazocine-induced backwards walking is mediated via an agonistic action of the drug with opioid kappa receptors. The data may contribute to the discussion concerning opioid kappa receptors and the psychotomimetic effects of some opioid analgesic drugs.     

痰热清注射液治疗小儿支气管肺炎的临床疗效研究

目的:观察痰热清注射液治疗小儿急性支气管肺炎的临床疗效及安全性。方法:将62例支气管肺炎患儿随机分为观察组和对照组各31例,两组均给予阿奇霉素治疗7天,在此基础上观察组另加痰热清注射液治疗。结果:观察组在退热时间、咳痰喘消失时间、肺部体征消失时间、住院天数均较对照组短,差异有统计学意义(均P<0.05);观察组总有效率为93.55%,对照组有效率为77.42%,两组比较差异有统计学意义(P<0.05);复发率观察组(2.94%)明显低于对照组(12.90%),差异有统计学意义(P<0.05);观察组无明显的不良反应发现。结论:痰热清注射液是治疗小儿支气管肺炎有效而安全的药物。     

痰热清注射液治疗肾综合征出血热疗效观察

目的观察痰热清注射液治疗肾综合征出血热(HFRS)早期患者的临床疗效。方法将100例发病5d以内单纯发热期的HFRS患者随机分为治疗组与对照组各50例。对照组给予以林格液为主的综合治疗,治疗组同时加用痰热清注射液20ml,静脉滴注,每日1次,疗程5d。结果在病毒血症的改善、减轻病情、越期、肾功能和肝功能恢复、血小板复常、减少并发症、治疗率和总有效率诸方面,治疗组均显著优于对照组(P<0.05~0.01)。结论痰热清注射液治疗HFRS早期(≤5d)患者,有良好效果。     

Adenovirus-mediated kallikrein gene delivery attenuates hypertension and protects against renal injury in deoxycorticosterone-salt rats

To demonstrate potential therapeutic effects of kallikrein gene delivery in salt-induced hypertension and renal diseases, we delivered adenovirus carrying the human tissue kallikrein gene (Ad.CMV-cHK) into deoxycorticosterone acetate (DOCA)-salt hypertensive rats. A single intravenous injection of Ad.CMV-cHK caused a delay in the rise of blood pressure that began 2 days post gene delivery and lasted for more than 23 days. A maximal blood pressure reduction of 50 mm Hg was observed in rats receiving kallikrein gene delivery, as compared to rats receiving adenovirus containing the luciferase gene (Ad.CMV-Luc) (172 +/- 5 vs. 222 +/- 13 mm Hg, n = 6, P < 0.01). Throughout the experimental period, a blood pressure reduction of at least 32 mm Hg was observed in the DOCA-salt rats injected with Ad.CMV-cHK as compared to DOCA-salt rats receiving control adenovirus. Immunoreactive human tissue kallikrein levels were detected in rat serum and urine post gene delivery. Adenovirus-mediated kallikrein gene delivery caused a significant reduction in urinary excretion, urinary protein levels and body weight. Morphological examination of the kidney showed that kallikrein gene transfer significantly reduced DOCA-salt-induced glomerular sclerotic lesions, brush border disruption of proximal tubules, tubular dilatation and protein cast accumulation. These findings showed that the expression of human tissue kallikrein via gene delivery has protective effects against hypertension and renal injury in DOCA-salt hypertensive rats.     

Effects of ingestion of chlorocholine chloride and cyclophosphamide on Venezuelan equine encephalitis virus infections in deer mice (Peromyscus maniculatus)

We investigated the effects of chlorocholine chloride (CCC), a plant growth regulator, and cyclophosphamide (CP), a known immunosuppressant, on the ability of deer mice (Peromyscus maniculatus) to resist challenge with a sublethal dose of Venezuelan equine encephalitis virus ( VEEV ). The toxicants were continuously delivered in low doses in the feed; CP at 20 mg/kg body wt/day and CCC at 1, 10, 20 or 40 mg/kg body wt/day. Mice were inoculated with 3 X 10(4) plaque forming units (pfu) of VEEV after eating experimental feed for 23 days. Mice were bled daily for 7 days and at selected intervals from 8 to 63 days post inoculation (PI) for viremia and antibody titer determinations. CP treatment increased the duration of viremia and significantly (P less than or equal to 0.05) decreased mean viremia titers. Antibody titers were significantly (P less than or equal to 0.05) depressed in CP-treated mice compared to controls. CP treatment increased mortality. CCC had no effect on viremia duration or titer except when given in doses of 1 mg/kg body wt/day when it significantly (P less than or equal to 0.05) decreased mean viremia titers compared to controls. Early antibody responses were increased by CCC treatment except in the 10 mg/kg body wt/day treatment group in which titers were decreased. By 30 days PI antibody titers of CCC-treated mice were no different from controls.     

Motor hypoactivity induced by neurotensin and related peptides in mice

The tridecapeptide neurotensin (NT) induces a variety of behavioral changes in animals. The present study characterizes the behavioral hypoactivity observed after intracerebroventricular (ICV) injection in mice. At doses higher than 25 ng, NT induced a reduction of general motor activity and increases in immobility which lasted for about one hour. The NT-related amphibian skin peptide xenopsin was about 70-fold more potent than NT itself. After repeated NT-injections, tolerance developed within 2-4 days and disappeared within 2-4 days after cessation of the treatment. The motor hypoactivity induced by NT was not attenuated by pretreatment with naloxone (5 mg/kg, SC). Furthermore, amphetamine-induced locomotor activity was not blocked by NT or xenopsin. These results suggest that the NT-effect is not mediated by a stimulation of opioid mechanisms or attenuation of dopamine-mediated events.     

雾化吸入布地奈德治疗慢性阻塞性肺疾病急性加重期的研究

目的观察短期雾化吸入布地奈德混悬液对慢性阻塞性肺疾病急性加重期（AECOPD）的疗效及安全性。方法126例AECOPD患者完全随机分为3组,每组42例,对照组给予常规的支气管扩张剂沙丁胺醇,静脉注射生理盐水100ml,1次／d;全身激素组在对照组的基础上静脉注射甲基泼尼松龙40mg,1次／d;吸入布地奈德组在支气管扩张剂的基础上给予雾化吸入布地奈德混悬液2mg,1次／8h。3组患者均于治疗前及治疗后24h、72h、7d及10d测定肺功能、动脉血气分析及空腹血糖等。结果在疗程结束后,共有6例退出,3组退出的人数分别为对照组3例,全身激素组1例,吸入布地奈德组2例,3组退出人数及退出原因差异无统计学意义。治疗后,3组肺功能及动脉血气较治疗前有明显改善（P〈0．05）,其中,全身激素组和吸入布地奈德组的肺功能及动脉血气指标较对照组恢复得更快;24h后,全身激素组PaO2,吸入布地奈德组PaO2、FEV。％的改善具有统计学意义,而对照组最早出现有统计学意义的改善在72h后。吸入布地奈德组与全身激素组除血糖外,其余各指标比较差异无统计学意义。结论吸入布地奈德混悬液对AECOPD患者是有效的,可替代使用全身激素治疗AECOPD。