The rabbit as an experimental model for regional chemotherapy. 2. Isolated perfusion of hindlimbs

Regional chemotherapy to a tumour is most commonly delivered by intra-arterial infusion. An alternative method of regional drug delivery, isolated perfusion, may be used where anatomical considerations permit. The technique of isolated perfusion in the rabbit hindlimb is described. The use of this model with the implantable rabbit VX2 carcinoma allows estimation of drug uptake by normal tissues, primary tumour and popliteal lymph node metastases. Correlation of such data with blood flow measurements enables targeting of new cytotoxic agents to be evaluated. The effect of perfusate composition on tissue uptake of such an agent, the plant toxin ricin, has been determined.     

Potent effects of aerosol compared with intravenous treprostinil on the pulmonary circulation.

Inhaled vasodilator therapy for pulmonary hypertension may decrease the systemic side effects commonly observed with systemic administration. Inhaled medications only reach ventilated areas of the lung, so local vasodilation may improve ventilation-perfusion matching and oxygenation. We compared the effects of intravenous vs. aerosolized treprostinil on pulmonary and systemic hemodynamics in an unanesthetized sheep model of sustained acute pulmonary hypertension. Acute, stable pulmonary hypertension was induced in instrumented unanesthetized sheep by infusing a PGH(2) analog, U-44069. The sheep were then administered identical doses of treprostinil either intravenously or by aerosol. Systemic and pulmonary hemodynamics were recorded during each administration. Both intravenous and aerosol delivery of treprostinil reduced pulmonary vascular resistance and pulmonary arterial pressure, but the effect was significantly greater with aerosol delivery (P < 0.05). Aerosol delivery of treprostinil had minimal effects on systemic hemodynamics, whereas intravenous delivery increased heart rate and cardiac output and decreased left atrial pressure and systemic blood pressure. Aerosol delivery of the prostacyclin analog treprostinil has a greater vasodilatory effect in the lung with minimal alterations in systemic hemodynamics compared with intravenous delivery of the drug. We speculate that this may result from treprostinil stimulated production of vasodilatory mediators from pulmonary epithelium.     

Group B Streptococcus-induced acidosis in newborn swine: regional oxygen transport and lactate flux.

To investigate the mechanism of metabolic acidosis resulting from group B streptococcal sepsis, oxygen metabolism and lactate flux of the cerebrum, hindlimb, liver, splanchnic organs, and systemic vascular bed as a whole were examined. Nine 3- to 5-day-old awake and spontaneously breathing piglets were studied before and after 3, 4, and 5 h of continuous live group B Streptococcus infusion. After 5 h, oxygen delivery was decreased to all organs and to the whole systemic vascular bed. Increased oxygen extraction compensated for reduced oxygen delivery in the liver and splanchnic organs; however, it only partially offset reduced oxygen delivery to the hindlimb and systemic vascular bed. Cerebral oxygen extraction did not increase. As a result, oxygen uptake was reduced in the cerebrum, hindlimb, and systemic vascular bed. At 5 h of bacterial infusion, arterial lactate concentration was increased with regional lactate efflux from the cerebrum and hindlimb and influx to the liver (P less than 0.05 vs. zero or no net flux). We conclude that group B Streptococcus-induced metabolic acidosis is associated with regional lactate efflux from vascular beds in which oxygen uptake is reduced. We speculate that the quantity of net lactate efflux from vascular beds with insufficient oxygen uptake exceeds the net influx into organs such as the liver, resulting in metabolic acidosis.     

Systemic p53 gene therapy of cancer with immunolipoplexes targeted by anti-transferrin receptor scFv.

BACKGROUND: A long-standing goal in genetic therapy for cancer is a systemic gene delivery system that selectively targets tumor cells, including metastases. Here we describe a novel cationic immunolipoplex system that shows high in vivo gene transfer efficiency and anti- tumor efficacy when used for systemic p53 gene therapy of cancer. MATERIALS AND METHODS: A cationic immunolipoplex incorporating a biosynthetically lipid-tagged, anti-transferrin receptor single-chain antibody (TfRscFv), was designed to target tumor cells both in vitro and in vivo. A human breast cancer metastasis model was employed to evaluate the in vivo efficacy of systemically administered, TfRscFv-immunolipoplex-mediated, p53 gene therapy in combination with docetaxel. RESULTS: The TfRscFv-targeting cationic immunolipoplex had a size of 60-100 nm, showed enhanced tumor cell binding, and improved targeted gene delivery and transfection efficiencies, both in vitro and in vivo. The p53 tumor suppressor gene was not only systemically delivered by the immunolipoplex to human tumor xenografts in nude mice but also functionally expressed. In the nude mouse breast cancer metastasis model, the combination of the p53 gene delivered by the systemic administration of the TfRscFv-immunolipoplex and docetaxel resulted in significantly improved efficacy with prolonged survival. CONCLUSIONS: This is the first report using scFv-targeting immunolipoplexes for systemic gene therapy. The TfRscFv has a number of advantages over the transferrin (Tf) molecule itself: (1) scFv has a much smaller size than Tf producing a smaller immunolipoplex giving better penetration into solid tumors; (2) unlike Tf, the scFv is a recombinant protein, not a blood product; (3) large scale production and strict quality control of the recombinant scFv, as well as scFv-immunolipoplex, are feasible. The sensitization of tumors to chemotherapy by this tumor-targeted and efficient p53 gene delivery method could lower the effective dose of the drug, correspondingly lessening the severe side effects, while decreasing the possibility of recurrence. Moreover, this approach is applicable to both primary and recurrent tumors, and more significantly, metastatic disease. The TfRscFv-targeting of cationic immunolipoplexes is a promising method of tumor targeted gene delivery that can be used for systemic gene therapy of cancer with the potential to critically impact the clinical management of cancer.     

Adipose-Derived Mesenchymal Stem Cell Administration Does Not Improve Corneal Graft Survival Outcome

The effect of local and systemic injections of mesenchymal stem cells derived from adipose tissue (AD-MSC) into rabbit models of corneal allograft rejection with either normal-risk or high-risk vascularized corneal beds was investigated. The models we present in this study are more similar to human corneal transplants than previously reported murine models. Our aim was to prevent transplant rejection and increase the length of graft survival. In the normal-risk transplant model, in contrast to our expectations, the injection of AD-MSC into the graft junction during surgery resulted in the induction of increased signs of inflammation such as corneal edema with increased thickness, and a higher level of infiltration of leukocytes. This process led to a lower survival of the graft compared with the sham-treated corneal transplants. In the high-risk transplant model, in which immune ocular privilege was undermined by the induction of neovascularization prior to graft surgery, we found the use of systemic rabbit AD-MSCs prior to surgery, during surgery, and at various time points after surgery resulted in a shorter survival of the graft compared with the non-treated corneal grafts. Based on our results, local or systemic treatment with AD-MSCs to prevent corneal rejection in rabbit corneal models at normal or high risk of rejection does not increase survival but rather can increase inflammation and neovascularization and break the innate ocular immune privilege. This result can be partially explained by the immunomarkers, lack of immunosuppressive ability and immunophenotypical secretion molecules characterization of AD-MSC used in this study. Parameters including the risk of rejection, the inflammatory/vascularization environment, the cell source, the time of injection, the immunosuppression, the number of cells, and the mode of delivery must be established before translating the possible benefits of the use of MSCs in corneal transplants to clinical practice.     

Multiscale models of the hybrid palliation for hypoplastic left heart syndrome

A less-invasive procedure that combines interventional stent placement in the ductus arteriosus and surgical banding of the branch pulmonary arteries has been recently introduced in the treatment of the hypoplastic left heart syndrome (HLHS). The hemodynamic behaviour of this hybrid approach has not been examined before in a mathematical model. In this study, a mathematical model of the hybrid procedure for HLHS is described, applying a multiscale approach that couples 3D models of the area of the surgical operation and lumped parameter models of the remaining circulation. The effects of various degrees of pulmonary banding and different stent sizes inserted in the ductus arteriosus on pulmonary-systemic flow ratio, cardiac output and oxygen delivery were assessed. Computational results suggest that balanced systemic and pulmonary blood flow and optimal systemic oxygen delivery are sensitive to the degree of pulmonary arterial banding and not to the size of the ductal stent.     

Safety and immunomodulatory effects of allogeneic canine adipose-derived mesenchymal stromal cells transplanted into the region of the lacrimal gland,the gland of the third eyelid and the knee joint

Background aimsMesenchymal stromal cells (MSCs) have been extensively studied as a cellular therapeutic for various pathologic conditions. However, there remains a paucity of data regarding regional and systemic safety of MSC transplantations, particularly with multiple deliveries of allogeneic cells. The purpose of this study was to investigate the safety and systemic immunomodulatory effects of repeated local delivery of allogeneic MSCs into the region of the lacrimal gland, the gland of the third eyelid and the knee joint in dogs.MethodsAllogeneic adipose tissue-derived canine MSCs were delivered to the regions of the lacrimal gland and the third eyelid gland as well as in the knee joints of six healthy laboratory beagles as follows: six times with 1-week intervals for delivery to the lacrimal gland and the third eyelid gland regions and three to four times with 1- to 2-week intervals for intra-articular transplantations. Dogs were sequentially evaluated by clinical examination. At the conclusion of the study, dogs were humanely euthanized, and a complete gross and histopathologic examination of all organ systems was performed. Mixed leukocyte reactions were also performed before the first transplantation and after the final transplantation.ResultsClinical and pathologic examinations found no severe consequences after repeated MSC transplantations. Results of mixed leukocyte reactions demonstrated suppression of T-cell proliferation after MSC transplantations.ConclusionsThis is the first study to demonstrate regional and systemic safety and systemic immunomodulatory effects of repeated local delivery of allogeneic MSCs in vivo.     

Regional intravascular sympathetic blockade for better results in flap surgery: an experimental study of free flaps, island flaps, and pedicle flaps in the rabbit ear

Flap survival is still a major problem in reconstructive surgery. Increased flap survival after systemic administration of drugs inhibiting the adrenergic system has been reported in experimental studies. The clinical use, however, is restricted by systemic side effects. It has been demonstrated that, using guanethidine, an effective regional intravascular sympathetic (RIS) block can be obtained without systemic effects. Using this type of block, an experimental study was made on the survival and quality of different types of flaps in the rabbit ear. The results obtained in 72 flaps created in the ears of 36 rabbits were assessed by the extent of flap edema, peripheral neovascularization, flap temperature, and flap surviving area. The RIS block reduced edema and scab formation, caused higher flap temperatures, better neovascularization, and increased surviving flap area, as compared with equal flaps in the untreated contralateral ear of the same animal. The effect of RIS block may be considered as a "pharmacological delay" procedure. From the experiments as well as clinical experience, it may be concluded that this technique is a safe and effective procedure. Therefore, the RIS block method is recommended for clinical use in flap techniques in extremities of man.     

In vivo rabbit hindquarter model for assessment of regional burn hypermetabolism.

Severe burn injury evokes hypermetabolism and muscle wasting, despite nominally adequate nutrition. Although there is much information on whole organism and isolated tissue metabolism after burn injury, data examining regional burn hypermetabolism in vivo are lacking. Using surgically implanted (general anesthesia) regional vascular catheters and primed constant infusion of l-[1-(13)C]phenylalanine tracer, we have determined in vivo burn-induced alterations in rabbit hindquarter protein and energy metabolism. Burn injury evokes increased whole body resting energy expenditure and phenylalanine turnover, accompanied by significantly increased hindquarter proteolysis, creating a negative protein balance in burned rabbit hindquarter. Hindquarter oxygen consumption showed an increase after burn injury, but it did not reach statistical significance. Burn-induced changes in hindquarter protein turnover account for approximately one-third of the whole animal hypermetabolism. This model offers a system for regional manipulation of postburn hypermetabolism.     

Regional hydrodynamic gene delivery to the rat liver with physiological volumes of DNA solution

BACKGROUND: The major barrier to the clinical application of hydrodynamic gene delivery to the liver is the large volume of fluid required using standard protocols. Regional hydrodynamic gene delivery via branches of the portal vein has not previously been reported, and we have evaluated this approach in a rat model. METHODS: The pGL3 plasmid with the luciferase reporter gene was used at 50 micro g/ml in isotonic solutions, and was administered with a syringe pump for precise control of the hydrodynamic conditions evaluated. Gene expression was individually measured in six anatomically distinct liver lobes. The effect of systemic chloroquine to promote endocytic escape and a (Lys)(16)-containing peptide to condense the DNA into approximately 100-nm nanoparticles was also evaluated. RESULTS: Hydrodynamic conditions for excellent gene delivery were obtained by using 3-ml volumes ( approximately 12 ml/kg) of isotonic DNA solution delivered at 24 ml/min to the right lateral lobe ( approximately 20% of the liver mass). Under these conditions, >95% of gene delivery usually occurred in the targeted right lateral lobe. Outflow obstruction was essential for gene delivery, both at optimal and at very low levels of hydrodynamic gene delivery. The use of systemic chloroquine to promote endocytic escape did not augment hydrodynamic gene delivery, while condensation of DNA in non-ionic isotonic solutions (5% dextrose) to nanoparticles of approximately 100 nm completely abolished gene delivery. CONCLUSIONS: Regional hydrodynamic gene delivery via a branch of the portal vein offers a physiological model of liver gene therapy, for experimental and clinical application.     

Modeling of the Norwood circulation: effects of shunt size, vascular resistances, and heart rate

Hypoplastic left heart syndrome is the most common lethal cardiac malformation of the newborn. Its treatment, apart from heart transplantation, is the Norwood operation. The initial procedure for this staged repair consists of reconstructing a circulation where a single outlet from the heart provides systemic perfusion and an interpositioning shunt contributes blood flow to the lungs. To better understand this unique physiology, a computational model of the Norwood circulation was constructed on the basis of compartmental analysis. Influences of shunt diameter, systemic and pulmonary vascular resistance, and heart rate on the cardiovascular dynamics and oxygenation were studied. Simulations showed that 1) larger shunts diverted an increased proportion of cardiac output to the lungs, away from systemic perfusion, resulting in poorer O2 delivery, 2) systemic vascular resistance exerted more effect on hemodynamics than pulmonary vascular resistance, 3) systemic arterial oxygenation was minimally influenced by heart rate changes, 4) there was a better correlation between venous O2 saturation and O2 delivery than between arterial O2 saturation and O2 delivery, and 5) a pulmonary-to-systemic blood flow ratio of 1 resulted in optimal O2 delivery in all physiological states and shunt sizes.     

Delivering healthy mitochondria for the therapy of mitochondrial diseases and beyond

Mitochondrial transfer has been demonstrated to a play a physiological role in the rescuing of mitochondrial DNA deficient cells by co-culture with human mesenchymal stem cells. The successful replacement of mitochondria using microinjection into the embryo has been revealed to improve embryo maturation. Evidence of mitochondrial transfer has been shown to minimize injury of the ischemic-reperfusion rabbit heart model. In this mini review, the therapeutic strategies of mitochondrial diseases based on the concept of mitochondrial transfer are illustrated, as well as a novel approach to peptide-mediated mitochondrial delivery. The possible mechanism of peptide-mediated mitochondrial delivery in the treatment of the myoclonic epilepsy and ragged-red fiber disease is summarized. Understanding the feasibility of mitochondrial manipulation in cells facilitates novel therapeutic skills in the future clinical practice of mitochondrial disorder.     

Hepatocyte-directed gene transfer in vivo leads to transient improvement of hypercholesterolemia in low density lipoprotein receptor-deficient rabbits.

Familial hypercholesterolemia is an inherited disease in humans, caused by a deficiency of low density lipoprotein (LDL) receptors, that we have used as a model for developing liver-directed gene therapies. Our strategy is to reconstitute hepatic LDL receptor expression in vivo by administering a DNA-protein complex that is capable of targeting the delivery of functional LDL receptor genes to hepatocytes. Infusion of this DNA-protein complex into the peripheral circulation of a rabbit animal model for familial hypercholesterolemia resulted in hepatocyte-specific gene transfer and a temporary amelioration of hypercholesterolemia. This noninvasive approach to gene therapy should have applications in the treatment of a wide spectrum of human diseases.     

Systemic siRNA delivery to leukocyte-implicated diseases

Short interfering RNA (siRNA), a small duplex of RNA fragment, has proved as an extremely useful research tool to interrogate gene functions in test tubes. However, the transformation of siRNAs from a functional genomic tool into a new therapeutic modality has been hindered by ineffective delivery methods for systemic administration. In this review, we will discuss the recent advances in formulating new delivery strategies that target siRNAs to specific cells following systemic administration. Special emphasis will be given to leukocytes, since siRNA delivery remains exceptionally challenging here due to the unavailability of effective delivery technologies. We will not only detail new platforms that utilize leukocyte integrins as receptor targets for siRNAs delivery, but also show how one of these strategies has been utilized for in vivo drug target validation of a novel anti-inflammatory target, cyclin D1, for inflammatory bowel diseases.     

Transdermal delivery of scopolamine by natural submicron injectors: in-vivo study in pig

Transdermal drug delivery has made a notable contribution to medical practice, but has yet to fully achieve its potential as an alternative to oral delivery and hypodermic injections. While transdermal delivery systems would appear to provide an attractive solution for local and systemic drug delivery, only a limited number of drugs can be delivered through the outer layer of the skin. The most difficult to deliver in this way are hydrophilic drugs. The aquatic phylum Cnidaria, which includes sea anemones, corals, jellyfish and hydra, is one of the most ancient multicellular phyla that possess stinging cells containing organelles (cnidocysts), comprising a sophisticated injection system. The apparatus is folded within collagenous microcapsules and upon activation injects a thin tubule that immediately penetrates the prey and delivers its contents. Here we show that this natural microscopic injection system can be adapted for systemic transdermal drug delivery once it is isolated from the cells and uploaded with the drug. Using a topically applied gel containing isolated natural sea anemone injectors and the muscarinic receptor antagonist scopolamine, we found that the formulated injectors could penetrate porcine skin and immediately deliver this hydrophilic drug. An in-vivo study in pigs demonstrated, for the first time, rapid systemic delivery of scopolamine, with T(max) of 30 minutes and C(max) 5 times higher than in controls treated topically with a scopolamine-containing gel without cnidocysts. The ability of the formulated natural injection system to penetrate a barrier as thick as the skin and systemically deliver an exogenous compound presents an intriguing and attractive alternative for hydrophilic transdermal drug delivery.     

Triggered release of ciprofloxacin from nanostructured agglomerated vesicles

Nanostructured agglomerated vesicles encapsulating ciprofloxacin were evaluated for modulated delivery from the lungs in a healthy rabbit model. An aliphatic disulfide crosslinker, cleavable by cysteine was used to form cross-links between nanosized liposomes to form the agglomerates. The blood levels of drug after pulmonary instillation of free ciprofloxacin, liposomal ciprofloxacin, and the agglomerated liposomes encapsulating ciprofloxacin were evaluated. The liposomes and agglomerated vesicles showed extended release of drug into the blood over 24 hours, while the free ciprofloxacin did not. The agglomerates also allowed modulation of the drug release rate upon the introduction of cysteine into the lungs post-drug instillation; the cysteine-cleavable agglomerates accelerated their drug release rate, indicated by an increased level of drug in the blood. This technology holds promise for the post-administration modulation of antibiotic release, for the prevention and treatment of pulmonary and systemic infections.     

Epicutaneous immunotherapy results in rapid allergen uptake by dendritic cells through intact skin and downregulates the allergen-specific response in sensitized mice

Epicutaneous immunotherapy onto intact skin has proved to be an efficient and safe alternative treatment of allergy in an animal model with various allergens and in children for cow's milk allergy. The aim of this study was to analyze the different steps of the immunological handling of the allergen when deposited on intact skin using an epicutaneous delivery system and its immune consequences in sensitized BALB/c mice. As expected, when applied on intact skin, OVA exhibits neither a passive passage through the skin nor any detectable systemic delivery. The current study demonstrates that, after a prolonged application on intact skin, OVA is taken up by dendritic cells in the superficial layers of the stratum corneum and transported, after internalization, to the draining lymph nodes, with variations according to the previous level of sensitization of the mice. When OVA is applied with the epicutaneous delivery system repeatedly, specific local and systemic responses are down-modulated in association with the induction of regulatory T cells. Besides providing new insights into skin function in the presence of allergens, this study indicates that the skin might have a tolerogenic role, at least when kept intact.     

A new solution for improving gene delivery

Intense research has focused on methods for overcoming the various barriers to DNA delivery while simultaneously reducing toxicity. Recently, Yuan and colleagues discovered a 'leakage route', whereby viral vectors escaped the injection site and entered the systemic circulation and contributed to the systemic toxicity in cancer gene therapy. They also demonstrated that alginate solution sealed this leak. Although their work focused on intratumoral injection of viral vectors, the findings can be extended to drug delivery in general, particularly DNA delivery.     

Low permeation of systemically administered human ß-endorphin into rabbit brain measured by radioimmunoassays differentiating human and rabbit ß-endorphin

Two antisera against human β-endorphin were generated in rabbits. They were found to differ largely in their specificities. One antiserum did not recognize rabbit β-endorphin. This antiserum was used to investigate the permeation of human β-endorphin into rabbit brain and cerebrospinal fluid after systemic injection of the synthetic peptide (50 μg/kg). Over a period of two hours, a low but significant permeation was found to occur only into the hypothalamus. All other brain areas remained below radioimmunoassay detection limits of 100 fmoles/g. Post-injectional cerebrospinal fluid concentrations of human β-endorphin showed very low values (90 fmoles/ml maximally). A regional distribution of rabbit brain β-endorphin, very similar to other species, was found using the antiserum which detected rabbit β-endorphin.     

Optimization of shunt placement for the Norwood surgery using multi-domain modeling

An idealized systemic-to-pulmonary shunt anatomy is parameterized and coupled to a closed loop, lumped parameter network (LPN) in a multidomain model of the Norwood surgical anatomy. The LPN approach is essential for obtaining information on global changes in cardiac output and oxygen delivery resulting from changes in local geometry and physiology. The LPN is fully coupled to a custom 3D finite element solver using a semi-implicit approach to model the heart and downstream circulation. This closed loop multidomain model is then integrated with a fully automated derivative-free optimization algorithm to obtain optimal shunt geometries with variable parameters of shunt diameter, anastomosis location, and angles. Three objective functions: (1) systemic; (2) coronary; and (3) combined systemic and coronary oxygen deliveries are maximized. Results show that a smaller shunt diameter with a distal shunt-brachiocephalic anastomosis is optimal for systemic oxygen delivery, whereas a more proximal anastomosis is optimal for coronary oxygen delivery and a shunt between these two anatomies is optimal for both systemic and coronary oxygen deliveries. Results are used to quantify the origin of blood flow going through the shunt and its relationship with shunt geometry. Results show that coronary artery flow is directly related to shunt position.