Injectable In Situ Forming Depot Systems for Long-Acting Contraception.

Up to date, no long-acting reversible contraceptive (LARC) is developed to be injectable through needles smaller than 18 G and can also provide contraception for more than 3 months after single injection. In this study, injectable polymeric in situ forming depot (ISD) systems are developed to have injectability through 21-23 G needles, and capability of sustained release of levonorgestrel (LNG) for at least 7 months in vitro and in vivo after single subcutaneous injection in rats. The systems are polymeric solutions composed of biodegradable poly(lactide-co-glycolide) and poly(lactic acid) polymers dissolved in a mixture of solvents like N-methyl-2-pyrrolidone and benzyl benzoate or triethyl citrate. LNG released from ISD systems successfully suppressed the estrous cycle of rats at plasma concentration above 0.35 ng mL-1 . At the end of the treatment, when LNG plasma concentration drops down to be nondetectable, predictable return of fertility is observed in rats. The designed ISD systems have great potential to be further developed into robust injectable LARCs that can be injected through a 21 G or smaller needle and achieve a variety of contraception durations with high patient compliance and low cost.

Development and validation of sensitive LC/MS/MS method for quantitative bioanalysis of levonorgestrel in rat plasma and application to pharmacokinetics study.

Rapid, sensitive, selective and accurate LC/MS/MS method was developed for quantitative determination of levonorgestrel (LNG) in rat plasma and further validated for specificity, linearity, accuracy, precision, sensitivity, matrix effect, recovery efficiency and stability. Liquid-liquid extraction procedure using hexane:ethyl acetate mixture at 80:20 v:v ratio was employed to efficiently extract LNG from rat plasma. Reversed phase Luna column C18(2) (50×2.0mm i.d., 3µM) installed on a AB SCIEX Triple Quad™ 4500 LC/MS/MS system was used to perform chromatographic separation. LNG was identified within 2min with high specificity. Linear calibration curve was drawn within 0.5-50ng·mL(-1) concentration range. The developed method was validated for intra-day and inter-day accuracy and precision whose values fell in the acceptable limits. Matrix effect was found to be minimal. Recovery efficiency at three quality control (QC) concentrations 0.5 (low), 5 (medium) and 50 (high) ng·mL(-1) was found to be >90%. Stability of LNG at various stages of experiment including storage, extraction and analysis was evaluated using QC samples, and the results showed that LNG was stable at all the conditions. This validated method was successfully used to study the pharmacokinetics of LNG in rats after SubQ injection, providing its applicability in relevant preclinical studies.

Long-acting injectable hormonal dosage forms for contraception.

Although great efforts have been made to develop long-acting injectable hormonal contraceptives for more than four decades, few long-acting injectable contraceptives have reached the pharmaceutical market or even entered clinical trials. On the other hand, in clinical practice there is an urgent need for injectable long-acting reversible contraceptives which can provide contraceptive protection for more than 3 months after one single injection. Availability of such products will offer great flexibility to women and resolve certain continuation issues currently occurring in clinics. Herein, we reviewed the strategies exploited in the past to develop injectable hormonal contraceptive dosages including drug microcrystal suspensions, drug-loaded microsphere suspensions and in situ forming depot systems for long-term contraception and discussed the potential solutions for remaining issues met in the previous development.

Intra-ophthalmic artery chemotherapy triggers vascular toxicity through endothelial cell inflammation and leukostasis.

Purpose. Super-selective intra-ophthalmic artery chemotherapy (SSIOAC) is an eye-targeted drug-delivery strategy to treat retinoblastoma, the most prevalent primary ocular malignancy in children. Unfortunately, recent clinical reports associate adverse vascular toxicities with SSIOAC using melphalan, the most commonly used chemotherapeutic. Methods. To explore reasons for the unexpected vascular toxicities, we examined the effects of melphalan, as well as carboplatin (another chemotherapeutic used with retinoblastoma), in vitro using primary human retinal endothelial cells, and in vivo using a non-human primate model, which allowed us to monitor the retina in real time during SSIOAC. Results. Both melphalan and carboplatin triggered human retinal endothelial cell migration, proliferation, apoptosis, and increased expression of adhesion proteins intracellullar adhesion molecule-1 [ICAM-1] and soluble chemotactic factors (IL-8). Melphalan increased monocytic adhesion to human retinal endothelial cells. Consistent with these in vitro findings, histopathology showed vessel wall endothelial cell changes, leukostasis, and vessel occlusion. Conclusions. These results reflect a direct interaction of chemotherapeutic drugs with both the vascular endothelium and monocytes. The vascular toxicity may be related to the pH, the pulsatile delivery, or the chemotherapeutic drugs used. Our long-term goal is to determine if changes in the drug of choice and/or delivery procedures will decrease vascular toxicity and lead to better eye-targeted treatment strategies.

Real-time ophthalmoscopic findings of superselective intraophthalmic artery chemotherapy in a nonhuman primate model.

OBJECTIVE: To report real-time ophthalmoscopic findings during superselective intraophthalmic artery chemotherapy (SSIOAC) in a nonhuman primate model. METHODS: Six adult male Rhesus macaques (Macacca mulatta) were randomly assigned to 1 of 2 treatment cohorts: melphalan (5 mg/30 mL) or carboplatin (30 mg/30 mL). Each animal underwent 3 separate SSIOAC procedures at 3-week intervals. Digital retinal images were obtained during each infusion. Intravenous fluorescein angiography was performed immediately after each procedure. RESULTS: All SSIOAC procedures were successfully completed. Toxicities were equally distributed between drug cohorts. Systemic toxicities included mild bone marrow suppression in all animals and anorexia in 1. One animal had greater than 50% narrowing of the treated ophthalmic artery after its second infusion. All 18 procedures (100%) resulted in pulsatile optic nerve and choroid blanching, retinal artery narrowing, and retinal edema. Of the 18 procedures, retinal artery sheathing was found during 17 (94%), and retinal artery precipitates were seen in 10 (56%); choroidal hypoperfusion was seen by fluorescein angiogram in 18 (100%). CONCLUSION: Real-time ophthalmic investigations are useful and, in our nonhuman primate model, indicate prevalent, acute ocular vascular toxicities during SSIOAC. CLINICAL RELEVANCE: Real-time retinal imaging is feasible in a nonhuman primate model of SSIOAC. Application to SSIOAC in children may shed insight into reported vascular toxicities.

Sensitivity of perianal tape impressions to diagnose pinworm (Syphacia spp.) infections in rats (Rattus norvegicus) and mice (Mus musculus).

We determined the sensitivity of perianal tape impressions to detect Syphacia spp. in rats and mice. We evaluated 300 rat and 200 mouse perianal impressions over 9 wk. Pinworm-positive perianal tape impressions from animals with worm burdens at necropsy were considered as true positives. Conversely, pinworm-negative perianal tape impressions from animals with worm burdens were considered false negatives. The sensitivity of perianal tape impressions for detecting Syphacia muris infections in rats was 100%, and for detecting Syphacia obvelata in mice was 85.5%. Intermittent shedding of Syphacia obvelata ova is the most probable explanation for the decreased sensitivity rate we observed in mice. We urge caution in use of perianal tape impressions alone for Syphacia spp. screening in sentinel mice and rats.

Effects of leukotriene inhibition on pulmonary morphology in rat pup lungs exposed to hyperoxia.

Assisted ventilation is necessary for treating preterm infants with respiratory distress syndrome. Unfortunately, high and prolonged concentrations of oxygen associated with assisted ventilation often lead to pulmonary changes, such as hemorrhage and inflammation. The resulting chronic pulmonary condition is known as bronchopulmonary dysplasia. Pulmonary changes characteristic of this syndrome can be produced in rat pups exposed to high oxygen levels. We exposed 21-d-old rats to room air or continuous 95% oxygen for 7 d and then allocated them into 6 groups to evaluate whether treatment with zileuton and zafirlukast, 2 agents which decrease the effects of leukotrienes, lessened the pulmonary effects of short-term hyperoxia. After 7 d, lung tissue was collected for light and electron microscopy. Pulmonary changes including edema, hemorrhage, alveolar macrophage influx, and Type II pneumocyte proliferation were graded on a numerical scoring system. Compared with controls exposed to hyperoxia [corrected] and saline, rats exposed to hyperoxia and treated with zileuton had significantly reduced levels of alveolar macrophage influx and Type II pneumocyte proliferation, but those exposed to hyperoxia [corrected] and treated with zafirlukast showed no significant reduction in any pulmonary changes. This study helps define pulmonary changes induced secondary to hyperoxia in rat pups and presents new information on the mechanisms of leukotriene inhibition in decreasing the severity of hyperoxic lung injury.

Skin basement membrane zone: a depository for circulating microbial antigen evoking psoriasis and autoimmunity.

BACKGROUND: Elevated levels of antibody to streptococcal exoenzymes have been found in patients with psoriasis or psoriatic arthritis. Research on the role of streptococcal antigen in psoriasis has been hampered by a potential molecular mimicry between streptococcal epitopes and human epidermal keratin. OBJECTIVE AND METHODS: Evidence of microbial product was sought in skin biopsies of psoriasis patients thought clinically to have either streptococcal carrier state or gastrointestinal candidal colonization. A polyclonal antibody to streptococcal-derived exoenzymes unlikely to share antigenic structures with normal human skin, and an anticandidal antibody, were used with linked streptavidin biotin amplification stain. RESULTS: The predicted microbial product appeared heavily in lesional epidermis, but unexpectedly also as a thin deposit along the skin basement membrane zone (SBMZ) of apparently unaffected skin. Staining was negative for nonpsoriatic subjects. CONCLUSIONS: The findings support a direct effect of microbial antigen in psoriasis. They also suggest an important role for SBMZ as a very large adhesive surface in the first step of a process of percutaneous epidermal elimination of foreign antigens and microbial toxins. The many autoimmune phenomena seen so often at the SBMZ are probably a physiologic part of this important immune function. Efforts to enhance the adhesive properties of SBMZ should be exploitable for both diagnostic and therapeutic benefit.

Posterior paresis and osteolysis in guinea pigs (Cavia porcellus) secondary to Freund's adjuvant immunization.

Bilateral hindlimb paresis occurred in 3 guinea pigs after immunization with an adjuvant-antigen mixture containing complete Freund's adjuvant. Doses were injected into unanaesthetized animals, divided among 3 or 4 sites, and given slightly off midline in the subcutaneous tissues of the back. Neurologic examination of affected animals revealed intact flexor and panniculus responses and limited voluntary movement of the hindlimbs. Histopathologic interpretation of 2 affected animals showed fibrogranulomatous material effacing the skeletal muscle and vertebral bone, with marked bone lysis and infiltration into the marrow space and spinal canal. In addition, multiple granulomas in the pulmonary parenchyma were noted. A postmortem radiograph of the excised thoracolumbar spine of 1 animal revealed a soft tissue swelling and "moth-eaten" and geographic osteolysis of 2 spinous processes. Hindlimb paresis and osteolysis likely resulted from accidental injection of the adjuvant-antigen mixture into the epaxial musculature and subsequent extension of injection site granulomas into the spinal canal.

Use of permethrin eradicated the tropical rat mite (Ornithonyssus bacoti) from a colony of mutagenized and transgenic mice.

The tropical rat mite, Ornithonyssus bacoti, was identified in a colony of mutagenized and transgenic mice at a large academic institution. O. bacoti is an obligate, blood-feeding ectoparasite with an extensive host range. Although the source of the infestation was likely feral rodents, none were found in the room housing infested mice. We hypothesize that construction on the floor above the vivarium and compromised ceiling integrity within the animal room provided for vermin entry and subsequent O. bacoti infestation. O. bacoti infestation was eliminated by environmental decontamination with synthetic pyrethroids and weekly application of 7.4% permethrin-impregnated cotton balls to mouse caging for five consecutive weeks. Visual examination of the macroenvironment, microenvironment, and colony for 38 days confirmed the efficacy of treatment. We noted no treatment-related toxicities or effects on colony production.

Toxic epidermal necrolysis in two rhesus macaques (Macaca mulatta) after administration of rituximab.

A 2- and a 7-year-old rhesus macaque developed toxic epidermal necrolysis (TEN) after administration of ritux imab. Rituximab, a chimeric monoclonal antibody (mAb) directed against the CD20 antigen on B lymphocytes, is used to treat certain B cell neoplasias. The macaques were part of a gene therapy study that involved administering an adeno-associated viral vector encoding human factor IX (hFIX) to the animals. Both animals developed antibody against hFIX, which eliminated expression of the protein. Rituximab was administered to deplete the population of B cells producing antibodies against the protein. Two days after treatment, the 7-year-old animal developed erythemic skin lesions that rapidly progressed in severity, resulting in epidermal sloughing and ulceration. Despite aggressive treatment with analgesics, antibiotics, and corticosteroids, the animal had to be euthanized 5 days later. The 2-year-old macaque had no reaction to the initial dose of rituximab and received a second infusion 2 weeks later. Two days after drug administration, skin lesions developed; aggressive analgesic, antibiotic, and corticosteroid treatment was initiated, and the lesions resolved. A third rituximab dose was given approximately 2 months after the second. Skin lesions developed and were treated. The animal made a full recovery. In both cases, skin biopsies were taken and histopathologic findings were consistent with TEN. A severe, life-threatening condition, TEN manifests as an intolerance reaction in the skin. The most common cause of TEN is a response to previous drug administration. To our knowledge, this condition has not been reported in association with rituximab administration in macaques.

Enrichment and nonhuman primates: "first, do no harm".

Since the 1998 publication of The Psychological Well-Being of Nonhuman Primates by the National Research Council, and the 1991 implementation of the 1985 Animal Welfare Act Amendment, many formal and informal nonhuman primate enrichment programs have been put into practice. Reports of their successes and failures, however, are few. All programs have at least two things in common: (1) They are best when designed and maintained by teams of individuals with species-specific expertise; (2) the members of those teams, the stakeholders, usually include principal investigators, animal care and use committee members, veterinarians, and animal care staff. Discussions in this article address general principles about enrichment, goals of such programs from the perspective of each of the major stakeholders, and recently published sources of related information. These discussions follow the central premise that enrichment should benefit all involved and "First, do no harm."

Chronic prenatal exposure to cocaine alters cerebrovascular responses in newborn pigs.

Maternal cocaine abuse may increase the incidence of perinatal asphyxia. In nonexposed asphyxiated neonates, decreased cerebrospinal fluid (CSF) cAMP concentrations are associated with poor neurological outcome. On the other hand, cocaine increases central nervous system (CNS) cAMP. Therefore, we hypothesized that in utero cocaine exposure may increase brain cAMP and thereby preserve cerebrovascular responses to cAMP-dependent stimuli following asphyxia. Pregnant pigs received either cocaine (1 mg/kg, i.v.) twice weekly during the last trimester or normal saline vehicle (sham-control) and were allowed to deliver vaginally at term. Cranial windows were implanted in the newborn pigs within the first week of life and used to collect CSF for cAMP determinations and to assess changes in pial arteriolar diameters (PAD). In the first part of the study, pial arteriolar responses to different vasodilator and vasoconstrictor stimuli were evaluated in piglets prior to asphyxia (n = 20). In newborn pigs exposed to cocaine, cerebrovascular responses to hypercapnia and norepinephrine were significantly exaggerated compared to controls. Then, piglets were randomly selected for the second part of the study that involved prolonged asphyxia (n = 12). In cocaine-exposed but not sham-control piglets, CSF cAMP increased markedly during asphyxia. In the sham piglets, but not the cocaine-exposed piglets, CSF cAMP fell progressively below the baseline during recovery. Cerebrovascular reactivity to cAMP-dependent stimuli (hypercapnia and isoproterenol) was preserved during recovery from asphyxia in the cocaine-exposed piglets but significantly attenuated in the sham controls. We conclude that piglets with chronic prenatal exposure to cocaine show exaggerated cerebrovascular responses to vasogenic stimuli and preserved cAMP-dependent cerebral vasoreactivity following asphyxia.

Activated charcoal and the absorption of ferrous sulfate in rats.

The treatment of iron poisoning has typically not included the administration of activated charcoal due to the lack of evidence supporting its efficacy. Several in vitro studies have demonstrated good adsorption of iron in a variety of pH ranges that were comparable to those found with other drugs for which activated charcoal is clinically used. This study was designed to determine whether activated charcoal altered the gastrointestinal absorption of toxic doses of iron as ferrous sulfate in an in vivo model. Seventy-five male Sprague-Dawley rats were randomly assigned into 5 groups: control given only distilled water; 100 mg elemental Iron and water; 1:1 charcoal to iron; 2:1 charcoal to iron; and 4:1 charcoal to iron. All treatments were administered consecutively by gavage within 5 min. Physiological measurements and blood samples were taken at 0, 1, 4 and 8 h after treatment. There were no consistent differences in physiological measurements among the 5 groups. Mean serum iron concentrations did not differ among Groups 2, 3, 4 and 5 at the 4 sampling times except at I h between Groups 4 and 5. The area under the curve for serum iron concentrations did not differ among the treatment groups. Activated charcoal did not alter the extent of iron absorption in the experimental model.

Sustained high-level expression of human factor IX (hFIX) after liver-targeted delivery of recombinant adeno-associated virus encoding the hFIX gene in rhesus macaques.

The feasibility, safety, and efficacy of liver-directed gene transfer was evaluated in 5 male macaques (aged 2.5 to 6.5 years) by using a recombinant adeno-associated viral (rAAV) vector (rAAV-2 CAGG-hFIX) that had previously mediated persistent therapeutic expression of human factor IX (hFIX; 6%-10% of physiologic levels) in murine models. A dose of 4 x 10(12) vector genomes (vgs)/kg of body weight was administered through the hepatic artery or portal vein. Persistence of the rAAV vgs as circular monomers and dimers and high-molecular-weight concatamers was documented in liver tissue by Southern blot analysis for periods of up to 1 year. Vector particles were present in plasma, urine, or saliva for several days after infusion (as shown by polymerase chain reaction analysis), and the vgs were detected in spleen tissue at low copy numbers. An enzyme-linked immunosorption assay capable of detecting between 1% and 25% of normal levels of hFIX in rhesus plasma was developed by using hyperimmune serum from a rhesus monkey that had received an adenoviral vector encoding hFIX. Two macaques having 3 and 40 rAAV genome equivalents/cell, respectively, in liver tissue had 4% and 8% of normal physiologic plasma levels of hFIX, respectively. A level of hFIX that was 3% of normal levels was transiently detected in one other macaque, which had a genome copy number of 25 before abrogation by a neutralizing antibody (inhibitor) to hFIX. This nonhuman-primate model will be useful in further evaluation and development of rAAV vectors for gene therapy of hemophilia B.

Consequences of maternal cocaine on cerebral microvascular functions in piglets.

Maternal cocaine abuse is associated with fetal and neonatal neurological abnormalities. Prolonged exposure to cocaine can induce blood flow disorders, growth restriction, and hypoxia in the newborn. We investigated the impact of chronic fetal cocaine exposure on cerebral microvascular reactivity and autonomic function in the piglets. Pregnant pigs received cocaine (1 mg/kg i.v.; twice weekly) or saline throughout the last trimester. Prenatal exposure to cocaine did not have any significant effect on the birth weight of the piglets as compared to the control. Following delivery, effects of recurrent prenatal cocaine exposure on cerebral microvascular functions were examined in piglets (3-6 days old). Pial arteriolar responses to applications of 5-hydroxytryptamine (5-HT), endothelin-1 (ET-1), and clonidine were examined using closed cranial windows. Functional effects of prenatal cocaine exposure on changes in mean arterial pressure (MAP) and pial arteriolar diameter induced by intracisternal injection (i.c.) of clonidine (1 microg/kg) were also determined. Topical applications of 5-HT, ET-1, and clonidine dose-dependently decreased pial arteriolar diameter in the control and these constrictions were significantly enhanced in the in utero cocaine-exposed piglets. Prenatal cocaine exposure did not have any significant effects on the resting MAP and heart rate as there were no differences between the groups. IC clonidine caused sustained decrease in MAP in both groups but the decrease was more pronounced in the cocaine than the control group. IC clonidine causes cerebral microvascular dilation coincident with the development of hypotension. Such dilation was severely attenuated in the cocaine group, even though the hypotension was much more pronounced than in the control. In conclusion, prenatal cocaine exposure resulted in attenuated autoregulatory vasodilation and potentiated responses to vasoconstrictor agents. The mechanisms behind the effects of in utero cocaine exposure on alteration of newborn cerebral functions need further investigation. Such actions may be important in development of cerebral pathologies associated with recurrent prenatal cocaine exposure.

Diarrhea in Neonatal Rats.

A litter of eight 10- to 12-day-old SPF Sprague-Dawley rat pups (Rattus norvegicus) was observed as runted, with rough, yellow-discolored hair coats and perineal scalding and soiling. The dam also had soft stool and perineal soiling. Both the dam and pups were active despite their unthrifty appearance. The clinical signs were consistent with diarrhea. Differential diagnoses for diarrhea in neonatal rats were considered, including bacterial, viral, and parasitic etiologies. The dam and pups were sacrificed, and the following diagnostic tests were performed: complete blood count, serology, necropsy, bacterial culture, and histopathology. Histopathology and culture results were consistent with a diagnosis of streptococcal enteropathy.