Biochemical and behavioural profile of NTBC treated Tyrosinemie type 1 mice.

BACKGROUND: Tyrosinemia type 1 (HT1) is a rare metabolic disorder caused by a defect in the tyrosine catabolic pathway. Since HT1 patients are treated with NTBC, outcome improved and life expectancy greatly increased. However extensive neurocognitive and behavioural problems have been described, which might be related to treatment with NTBC, the biochemical changes induced by NTBC, or metabolites accumulating due to the enzymatic defect characterizing the disease. OBJECTIVE: To study the possible pathophysiological mechanisms of brain dysfunction in HT1, we assessed blood and brain LNAA, and brain monoamine neurotransmitter metabolite levels in relation to behavioural and cognitive performance of HT1 mice. DESIGN: C57BL/6 littermates were divided in three different experimental groups: HT1, heterozygous and wild-type mice (n = 10; 5 male). All groups were treated with NTBC and underwent cognitive and behavioural testing. One week after behavioural testing, blood and brain material were collected to measure amino acid profiles and brain monoaminergic neurotransmitter levels. RESULTS: Irrespective of the genetic background, NTBC treatment resulted in a clear increase in brain tyrosine levels, whereas all other brain LNAA levels tended to be lower than their reference values. Despite these changes in blood and brain biochemistry, no significant differences in brain monoamine neurotransmitter (metabolites) were found and all mice showed normal behaviour and learning and memory. CONCLUSION: Despite the biochemical changes, NTBC and genotype of the mice were not associated with poorer behavioural and cognitive function of the mice. Further research involving dietary treatment of FAH-/- are warranted to investigate whether this reveals the cognitive impairments that have been seen in treated HT1 patients.

Modeling the cognitive effects of diet discontinuation in adults with phenylketonuria (PKU) using pegvaliase therapy in PAH-deficient mice.

Existing phenylalanine hydroxylase (PAH)-deficient mice strains are useful models of untreated or late-treated human phenylketonuria (PKU), as most contemporary therapies can only be initiated after weaning and the pups have already suffered irreversible consequences of chronic hyperphenylalaninemia (HPA) during early brain development. Therefore, we sought to evaluate whether enzyme substitution therapy with pegvaliase initiated near birth and administered repetitively to C57Bl/6-Pahenu2/enu2 mice would prevent HPA-related behavioral and cognitive deficits and form a model for early-treated PKU. The main results of three reported experiments are: 1) lifelong weekly pegvaliase treatment prevented the cognitive deficits associated with HPA in contrast to persisting deficits in mice treated with pegvaliase only as adults. 2) Cognitive deficits reappear in mice treated with weekly pegvaliase from birth but in which pegvaliase is discontinued at 3 months age. 3) Twice weekly pegvaliase injection also prevented cognitive deficits but again cognitive deficits emerged in early-treated animals following discontinuation of pegvaliase treatment during adulthood, particularly in females. In all studies, pegvaliase treatment was associated with complete correction of brain monoamine neurotransmitter content and with improved overall growth of the mice as measured by body weight. Mean total brain weight however remained low in all PAH deficient mice regardless of treatment. Application of enzyme substitution therapy with pegvaliase, initiated near birth and continued into adulthood, to PAH-deficient Pahenu2/enu2 mice models contemporary early-treated human PKU. This model will be useful for exploring the differential pathophysiologic effects of HPA at different developmental stages of the murine brain.

A novel Pah-exon1 deleted murine model of phenylalanine hydroxylase (PAH) deficiency.

Phenylalanine hydroxylase (PAH) deficiency, colloquially known as phenylketonuria (PKU), is among the most common inborn errors of metabolism and in the past decade has become a target for the development of novel therapeutics such as gene therapy. PAH deficient mouse models have been key to new treatment development, but all prior existing models natively express liver PAH polypeptide as inactive or partially active PAH monomers, which complicates the experimental assessment of protein expression following therapeutic gene, mRNA, protein, or cell transfer. The mutant PAH monomers are able to form hetero-tetramers with and inhibit the overall holoenzyme activity of wild type PAH monomers produced from a therapeutic vector. Preclinical therapeutic studies would benefit from a PKU model that completely lacks both PAH activity and protein expression in liver. In this study, we employed CRISPR/Cas9-mediated gene editing in fertilized mouse embryos to generate a novel mouse model that lacks exon 1 of the Pah gene. Mice that are homozygous for the Pah exon 1 deletion are viable, severely hyperphenylalaninemic, accurately replicate phenotypic features of untreated human classical PKU and lack any detectable liver PAH activity or protein. This model of classical PKU is ideal for further development of gene and cell biologics to treat PKU.

Cardiac tissue citric acid cycle intermediates in exercised very long-chain acyl-CoA dehydrogenase-deficient mice fed triheptanoin or medium-chain triglyceride.

Anaplerotic odd-chain fatty acid supplementation has been suggested as an approach to replenish citric acid cycle intermediate (CACi) pools and facilitate adenosine triphosphate (ATP) production in subjects with long-chain fatty acid oxidation disorders, but the evidence that cellular CACi depletion exists and that repletion occurs following anaplerotic substrate supplementation is limited. We exercised very long-chain acyl-CoA dehydrogenase-deficient (VLCAD-/-) and wild-type (WT) mice to exhaustion and collected cardiac tissue for measurement of CACi by targeted metabolomics. In a second experimental group, VLCAD-/- and WT mice that had been fed chow prepared with either medium-chain triglyceride (MCT) oil or triheptanoin for 4 weeks were exercised for 60 minutes. VLCAD-/- mice exhibited lower succinate in cardiac muscle at exhaustion than WT mice suggesting lower CACi in VLCAD-/- with prolonged exercise. In mice fed either MCT or triheptanoin, succinate and malate were greater in VLCAD-/- mice fed triheptanoin compared to VLCAD-/- animals fed MCT but lower than WT mice fed triheptanoin. Long-chain odd acylcarnitines such as C19 were elevated in VLCAD-/- and WT mice fed triheptanoin suggesting some elongation of the heptanoate, but it is unknown what proportion of heptanoate was oxidized vs elongated. Prolonged exercise was associated with decreased cardiac muscle succinate in VLCAD-/- mice in comparison to WT mice. VLCAD-/- fed triheptanoin had increased succinate compared to VLCAD-/- mice fed MCT but lower than WT mice fed triheptanoin. Cardiac CACi were higher following dietary ingestion of an anaplerotic substrate, triheptanoin, in comparison to MCT.

Blood phenylalanine reduction corrects CNS dopamine and serotonin deficiencies and partially improves behavioral performance in adult phenylketonuric mice.

Central nervous system (CNS) deficiencies of the monoamine neurotransmitters dopamine and serotonin have been implicated in the pathophysiology of neuropsychiatric dysfunction in human phenylketonuria (PKU). In this study, we confirmed the occurrence of brain dopamine and serotonin deficiencies in association with severe behavioral alterations and cognitive impairments in hyperphenylalaninemic C57BL/6-Pahenu2/enu2 mice, a model of human PKU. Phenylalanine-reducing treatments, including either dietary phenylalanine restriction or liver-directed gene therapy, initiated during adulthood were associated with increased brain monoamine content along with improvements in nesting behavior but without a change in the severe cognitive deficits exhibited by these mice. At euthanasia, there was in Pahenu2/enu2 brain a significant reduction in the protein abundance and maximally stimulated activities of tyrosine hydroxylase (TH) and tryptophan hydroxylase 2 (TPH2), the rate limiting enzymes catalyzing neuronal dopamine and serotonin synthesis respectively, in comparison to levels seen in wild type brain. Phenylalanine-reducing treatments initiated during adulthood did not affect brain TH or TPH2 content or maximal activity. Despite this apparent fixed deficit in striatal TH and TPH2 activities, initiation of phenylalanine-reducing treatments yielded substantial correction of brain monoamine neurotransmitter content, suggesting that phenylalanine-mediated competitive inhibition of already constitutively reduced TH and TPH2 activities is the primary cause of brain monoamine deficiency in Pahenu2 mouse brain. We propose that CNS monoamine deficiency may be the cause of the partially reversible adverse behavioral effects associated with chronic HPA in Pahenu2 mice, but that phenylalanine-reducing treatments initiated during adulthood are unable to correct the neuropathology and attendant cognitive deficits that develop during juvenile life in late-treated Pahenu2/enu2 mice.

Tetrahydrobiopterin treatment reduces brain L-Phe but only partially improves serotonin in hyperphenylalaninemic ENU1/2 mice.

Hyperphenylalaninemia (HPA) caused by hepatic phenylalanine hydroxylase (PAH) deficiency has severe consequences on brain monoamine neurotransmitter metabolism. We have studied monoamine neurotransmitter status and the effect of tetrahydrobiopterin (BH4) treatment in Pahenu1/enu2 (ENU1/2) mice, a model of partial PAH deficiency. These mice exhibit elevated blood L-phenylalanine (L-Phe) concentrations similar to that of mild hyperphenylalaninemia (HPA), but brain levels of L-Phe are still ~5-fold elevated compared to wild-type. We found that brain L-tyrosine, L-tryptophan, BH4 cofactor and catecholamine concentrations, and brain tyrosine hydroxylase (TH) activity were normal in these mice but that brain serotonin, 5-hydroxyindolacetic acid (5HIAA) and 3-methoxy-4-hydroxyphenylglycol (MHPG) content, and brain TH protein, as well as tryptophan hydroxylase type 2 (TPH2) protein levels and activity were reduced in comparison to wild-type mice. Parenteral L-Phe loading conditions did not lead to significant changes in brain neurometabolite concentrations. Remarkably, enteral BH4 treatment, which normalized brain L-Phe levels in ENU1/2 mice, lead to only partial recovery of brain serotonin and 5HIAA concentrations. Furthermore, indirect evidence indicated that the GTP cyclohydrolase I (GTPCH) feedback regulatory protein (GFRP) complex may be a sensor for brain L-Phe elevation to ameliorate the toxic effects of HPA. We conclude that BH4 treatment of HPA toward systemic L-Phe lowering reverses elevated brain L-Phe content but the recovery of TPH2 protein and activity as well as serotonin levels is suboptimal, indicating that patients with mild HPA and mood problems (depression or anxiety) treated with the current diet may benefit from supplementation with BH4 and 5-OH-tryptophan.

High dose sapropterin dihydrochloride therapy improves monoamine neurotransmitter turnover in murine phenylketonuria (PKU).

Central nervous system (CNS) deficiencies of the monoamine neurotransmitters, dopamine and serotonin, have been implicated in the pathophysiology of neuropsychiatric dysfunction in phenylketonuria (PKU). Increased brain phenylalanine concentration likely competitively inhibits the activities of tyrosine hydroxylase (TH) and tryptophan hydroxylase (TPH), the rate limiting steps in dopamine and serotonin synthesis respectively. Tetrahydrobiopterin (BH4) is a required cofactor for TH and TPH activity. Our hypothesis was that treatment of hyperphenylalaninemic Pah(enu2/enu2) mice, a model of human PKU, with sapropterin dihydrochloride, a synthetic form of BH4, would stimulate TH and TPH activities leading to improved dopamine and serotonin synthesis despite persistently elevated brain phenylalanine. Sapropterin (20, 40, or 100mg/kg body weight in 1% ascorbic acid) was administered daily for 4 days by oral gavage to Pah(enu2/enu2) mice followed by measurement of brain biopterin, phenylalanine, tyrosine, tryptophan and monoamine neurotransmitter content. A significant increase in brain biopterin content was detected only in mice that had received the highest sapropterin dose, 100mg/kg. Blood and brain phenylalanine concentrations were unchanged by sapropterin therapy. Sapropterin therapy also did not alter the absolute amounts of dopamine and serotonin in brain but was associated with increased homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA), dopamine and serotonin metabolites respectively, in both wild type and Pah(enu2/enu2) mice. Oral sapropterin therapy likely does not directly affect central nervous system monoamine synthesis in either wild type or hyperphenylalaninemic mice but may stimulate synaptic neurotransmitter release and subsequent metabolism.

Pharmacologic inhibition of L-tyrosine degradation ameliorates cerebral dopamine deficiency in murine phenylketonuria (PKU).

Monoamine neurotransmitter deficiency has been implicated in the etiology of neuropsychiatric symptoms associated with chronic hyperphenylalaninemia in phenylketonuria (PKU). Two proposed explanations for neurotransmitter deficiency in PKU include first, that chronically elevated blood L-phenylalanine (Phe) inhibits the transport of L-tyrosine (Tyr) and L-tryptophan (Trp), the substrates for dopamine and serotonin synthesis respectively, into brain. In the second hypothesis, elevated Phe competitively inhibits brain tyrosine hydroxylase (TH) and tryptophan hydroxylase (TPH) activities, the rate limiting steps in dopamine and serotonin synthesis. Dietary supplementation with large neutral amino acids (LNAA) including Tyr and Trp has been recommended for individuals with chronically elevated blood Phe in an attempt to restore amino acid and monoamine homeostasis in brain. As a potential alternative treatment approach, we demonstrate that pharmacologic inhibition of Tyr degradation through oral administration of nitisinone (NTBC) yielded sustained increases in blood and brain Tyr, decreased blood and brain Phe, and consequently increased dopamine synthesis in a murine model of PKU. Our results suggest that Phe-mediated inhibition of TH activity is the likely mechanism of impaired dopamine synthesis in PKU. Pharmacologic inhibition of Tyr degradation may be a promising adjunct therapy for CNS monoamine neurotransmitter deficiency in hyperphenylalaninemic individuals with PKU.

Hepatocytes from wild-type or heterozygous donors are equally effective in achieving successful therapeutic liver repopulation in murine phenylketonuria (PKU).

Successful restoration of phenylalanine (Phe) clearance following liver-directed gene therapy in murine phenylketonuria (PKU) is likely dependent upon both the number of cells successfully transduced and the amount of phenylalanine hydroxylase (PAH) activity expressed per cell. At low levels of transduction, Phe clearance could be limited by the low absolute number of PAH-expressing cells rather than the total amount of PAH activity produced in the liver. We have evaluated the interrelationship between the number of PAH positive cells, the amount of PAH activity produced and Phe clearance through experiments with hepatocyte-mediated therapeutic liver repopulation in the Pah(enu2) mouse, a model of PKU. We compared the therapeutic efficacy of transplantation with either wild-type hepatocytes or hepatocytes from heterozygous Pah(enu2/+) donors into PAH deficient, hyperphenylalaninemic Pah(enu2)/Pah(enu2) mice. The recipient mice were also homozygous for fumarylacetoacetate hydrolase (FAH) deficiency. In this model system, FAH positive donor hepatocytes enjoy a selective growth advantage in the FAH-deficient recipient. If Phe clearance is governed predominantly by the total PAH activity, then more heterozygous cells, which express lower PAH activity than wild-type cells, should be required to correct Phe clearance. If the absolute donor cell number is more important, then wild-type hepatocytes should have no advantage over heterozygous cells. We successfully carried out therapeutic liver repopulation with heterozygous donor cells in fifteen mice and an additional thirteen transplants with wild-type cells. Blood Phe was successfully reduced in both transplant groups, and the relationship between the final blood Phe level and the extent of liver repopulation with donor cells did not differ between the two donor groups. Regardless of the type of donor cell, liver repopulation of approximately 3-10% was sufficient to at least partially reduce blood phenylalanine, and blood Phe levels were completely corrected in mice that had attained greater than approximately 10% liver repopulation. We conclude from our study that the absolute number of PAH-expressing cells likely governs Phe clearance at least at the levels of repopulation reported here and that the amount of PAH activity per donor cell is a less critical variable. The implication for liver-directed gene therapy of PKU is that only partial correction of cellular PAH deficiency may yet improve Phe clearance as long as a sufficient number of hepatocytes is successfully transduced.

Therapeutic liver repopulation by transient acetaminophen selection of gene-modified hepatocytes.

Gene therapy by integrating vectors is promising for monogenic liver diseases, especially in children where episomal vectors remain transient. However, reaching the therapeutic threshold with genome-integrating vectors is challenging. Therefore, we developed a method to expand hepatocytes bearing therapeutic transgenes. The common fever medicine acetaminophen becomes hepatotoxic via cytochrome p450 metabolism. Lentiviral vectors with transgenes linked in cis to a Cypor shRNA were administered to neonatal mice. Hepatocytes lacking the essential cofactor of Cyp enzymes, NADPH-cytochrome p450 reductase (Cypor), were selected in vivo by acetaminophen administration, replacing up to 50% of the hepatic mass. Acetaminophen treatment of the mice resulted in over 30-fold expansion of transgene-bearing hepatocytes and achieved therapeutic thresholds in hemophilia B and phenylketonuria. We conclude that therapeutically modified hepatocytes can be selected safely and efficiently in preclinical models with a transient regimen of moderately hepatotoxic acetaminophen.

Development of a porcine model of phenylketonuria with a humanized R408W mutation for gene editing.

Phenylketonuria (PKU) is a metabolic disorder whereby phenylalanine metabolism is deficient due to allelic variations in the gene for phenylalanine hydroxylase (PAH). There is no cure for PKU other than orthotopic liver transplantation, and the standard of care for patients is limited to dietary restrictions and key amino acid supplementation. Therefore, Pah was edited in pig fibroblasts for the generation of PKU clone piglets that harbor a common and severe human mutation, R408W. Additionally, the proximal region to the mutation was further humanized by introducing 5 single nucleotide polymorphisms (SNPs) to allow for development of gene editing machinery that could be translated directly from the pig model to human PKU patients that harbor at least one classic R408W allele. Resulting piglets were hypopigmented (a single Ossabaw piglet) and had low birthweight (all piglets). The piglets had similar levels of PAH expression, but no detectable enzymatic activity, consistent with the human phenotype. The piglets were fragile and required extensive neonatal care to prevent failure to thrive and early demise. Phenylalanine levels rose sharply when dietary Phe was unrestricted but could be rapidly reduced with a low Phe diet. Fibroblasts isolated from R408W piglets show susceptibility to correction using CRISPR or TALEN, with subsequent homology-directed recombination to correct Pah. This pig model of PKU provides a powerful new tool for development of all classes of therapeutic candidates to treat or cure PKU, as well as unique value for proof-of-concept studies for in vivo human gene editing platforms in the context of this humanized PKU allele.

AAV-Mediated CRISPR/Cas9 Gene Editing in Murine Phenylketonuria.

Phenylketonuria (PKU) due to recessively inherited phenylalanine hydroxylase (PAH) deficiency results in hyperphenylalaninemia, which is toxic to the central nervous system. Restriction of dietary phenylalanine intake remains the standard of PKU care and prevents the major neurologic manifestations of the disease, yet shortcomings of dietary therapy remain, including poor adherence to a difficult and unpalatable diet, an increased incidence of neuropsychiatric illness, and imperfect neurocognitive outcomes. Gene therapy for PKU is a promising novel approach to promote lifelong neurological protection while allowing unrestricted dietary phenylalanine intake. In this study, liver-tropic recombinant AAV2/8 vectors were used to deliver CRISPR/Cas9 machinery and facilitate correction of the Pah enu2 allele by homologous recombination. Additionally, a non-homologous end joining (NHEJ) inhibitor, vanillin, was co-administered with the viral drug to promote homology-directed repair (HDR) with the AAV-provided repair template. This combinatorial drug administration allowed for lifelong, permanent correction of the Pah enu2 allele in a portion of treated hepatocytes of mice with PKU, yielding partial restoration of liver PAH activity, substantial reduction of blood phenylalanine, and prevention of maternal PKU effects during breeding. This work reveals that CRISPR/Cas9 gene editing is a promising tool for permanent PKU gene editing.

Reduction of rib fractures with a bioresorbable plating system: preliminary observations.

BACKGROUND: Operative fixation of rib fractures can reduce morbidity and mortality. Currently, resorbable fixation devices are used in a variety of surgical procedures. METHODS: A standard osteotomy was prepared in 30 New Zealand white rabbits at the 12th rib. Eighteen had surgical repair with bioresorbable plates and 12 underwent nonoperative management. Half the animals in each group were killed at 3-week postfracture and the remaining animals were killed at 6-week postfracture. Ribs were radiographed and processed histologically to assess fracture healing. Rib reduction was defined as the alignment of the rib ends in a structural condition similar to the prefractured state and quantitative radiomorphometry measured the radiopaque callus surrounding the rib injury sites. Statistical analysis was performed using Fisher's exact test and an unpaired Student's t test and significance was established at p < 0.05. RESULTS: At both the 3- and 6-week intervals, seven of the nine rib fractures remained reduced in the operative group, whereas zero of six and three of six of the rib fractures remained reduced, respectively, in the nonoperative group. A statistically significant increase in radiopaque callus surrounding the rib injury sites was observed at 3 and 6 weeks in the fixed groups. CONCLUSIONS: Fixation of rib fractures with a bioresorbable miniplate system was superior to nonoperative treatment at the 3-week interval, with a statistically significant increase in radiopaque callus formation at both 3 and 6 weeks. Additional studies will evaluate the biomechanical outcomes and degradation tissue response after extended in vivo intervals.

Revascularization of rat fasciocutaneous flap using CROSSEAL with VEGF protein or plasmid DNA expressing VEGF.

BACKGROUND: Fasciocutaneous tissue transfer is a common reconstructive procedure. Revascularization of flap tissue is an important component of tissue healing. Gene therapy offers an avenue through which the period of pedicle vascular dependency can be reduced. MATERIALS AND METHODS: Rat fasciocutaneous flaps were elevated and a two-hour ischemic time induced. Polycation complex (jet PEI) and human fibrin sealant CROSSEAL was applied between flap and underlying abdominal tissues. Group 1 (six rats) was the control; Group 2 (seven rats) had vascular endothelial growth factor (VEGF) protein applied; Group 3 (seven rats) had plasmid DNA expressing VEGF applied. Vascular pedicles were ligated on postoperative day 5, percentage flap survival evaluated on day 7. RESULTS: All flaps survived initial ischemia. Mean +/- SD percentage area of the flap that survived was 28.1 +/- 12.4 (Group 1), 71.6 +/- 16.2 (Group 2), and 77.5 +/- 12.7 (Group 3) (P < 0.001, Group 1-3, 2-3). No differences were observed between Groups 2 and 3. CONCLUSIONS: Locally administered VEGF protein or plasmid DNA expressing VEGF enhanced survival of fasciocutaneous flaps.

Osteogenic response of bone marrow stromal cells from normal and ovariectomized rats treated with a low dose of basic fibroblast growth factor.

Basic fibroblast growth factor (bFGF) is a potent mitogen that exhibits stimulatory effects on bone tissue regeneration. To gain further insight into the potential of bFGF for systemic therapy in osteoporosis, we investigated the responsiveness of bone marrow stromal cells (BMSCs) explanted from 7-month-old normal and ovariectomized (OVX) rats that were intravenously treated with a low dose of bFGF (25 microg/kg) for 2 weeks. The BMSCs were obtained using femoral aspiration and maintained in an osteogenic medium. The amount of cells recovered from bFGF-treated rats was lower than that from saline-treated rats, and proliferation of the cells was markedly less for the bFGF-treated rats. The BMSCs from the bFGF-treated rats also showed lower levels of specific alkaline phosphatase (ALP) activity (ALP/deoxyribonucleic acid) and mineralization. Expression of the extracellular matrix proteins critical for mineralization, in particular osteopontin, was greater for bFGF-treated cells from both types of animals in the first week of culture, after which the expression of all markers significantly declined. Dual energy x-ray absorptiometry analyses of the tibiae showed an increase in bone mineral density after bFGF treatment only for OVX rats. We conclude that osteoprogenitor cells were depleted from the marrow of bFGF-treated rats, most likely because of the stimulatory effect of bFGF on bone formation.

Managing chronic pain with encapsulated cell implants releasing catecholamines and endogenous opiods.

Spinal injections (intrathecal) of norepinephrine and/or opiod agonists are antinociceptive and when administered together may act in synergy. Spinal implants of adrenal chromaffin cells are an effective method for sustained delivery of the analgesic substances norepinephrine and enkephalin to the central nervous system (CNS). One method of packaging and implanting cell-loaded devices into the intrathecal space of recipients is by encapsulating the cell suspensions in a polymer membrane prior to implantation. Cells/tissue packaged within an encapsulating membrane obviate the need for immunosuppressive therapies in transplant recipients. In addition, device output can be quantified prior to implantation, and following the removal of the spinal implant. The ability to retrieve the devices with the present tubular configuration also confers an additional margin of safety over unencapsulated chromaffin cell implants. This paper reviews the research and clinical observations of cellular transplants containing adrenal chromaffin cells for relieving chronic pain. Encapsulated cell technology is discussed with an emphasis on our experiences developing pain-modulating clinical devices. The human-sized prototype devices were loaded with enzymatically isolated bovine chromaffin cells and maintained in vitro for 7 - 8 days in serum-free media. Two days prior to implantation, each device was assayed by static incubation to measure catecholamine and met-enkephalin output, and qualified devices (n = 6) were implanted into the sheep subarachnoid space for 6 weeks. Following a 6 week in life period, the retrieval forces of prototype devices were measured during removal from the subarachnoid space. Static incubation of the devices immediately following retrieval and after a 24 hour re-incubation period were used to quantify norepinephrine and met-enkephalin secretion profiles. This study demonstrated the safety, retrievability and maintenance of pharmacologically active encapsulated chromaffin cell-loaded devices with human implant dimensions.

Bone marrow cells from normal and ovariectomized rats respond differently to basic fibroblast growth factor and bone morphogenetic protein 2 treatment in vitro.

The protein growth factors basic fibroblast growth factor (bFGF) and bone morphogenetic protein 2 (BMP-2) are being actively pursued for bone tissue engineering. Although both proteins are capable of stimulating osteogenic activity of bone marrow cells (BMCs), no studies have addressed the effect of estrogen deficiency on the growth factor responsiveness of BMCs. This study investigated the osteogenic response of BMCs from normal and ovariectomized (OVX) rats to bFGF and BMP- 2. In the absence of growth factors, a higher number of total colony-forming units (t-CFU) and alkaline phosphatase-expressing CFU (ALP-CFU) were obtained with BMCs derived from OVX rats. The percentage of ALP-CFU, however, was not significantly different between BMCs from the two groups of rats. Whereas BMP-2 did not influence the t-CFU and percentage of ALP-CFU, bFGF decreased t-CFU in BMCs derived from OVX rats and reduced the percentage of ALP-CFU in BMCs from both types of rats. Consistent with the higher t-CFU, the number of mineralized colonies (min-CFU) was also higher for BMCs derived from OVX rats. The number of min-CFU was not influenced by BMP-2 treatment, but was reduced with bFGF treatment. Comparison of the growth factor effects on a per-cell (DNA) basis confirmed the expected stimulatory effect of BMP-2 on ALP activity and mineralization in BMCs from normal rats, but these two parameters were not unequivocally stimulated in BMCs from OVX rats. We conclude that BMCs derived from normal and OVX rats exhibited significant differences in their osteogenic response to bFGF and BMP-2 treatment.

Synthetic thermoreversible polymers are compatible with osteoinductive activity of recombinant human bone morphogenetic protein 2.

Recombinant human bone morphogenetic protein 2 (rhBMP-2) is currently in clinical studies as part of an implantable device that contains a biomaterial carrier. Implant retention of rhBMP-2 by the biomaterial carrier is important for the osteoinductive activity. To control in situ retention of rhBMP-2, thermoreversible polymers were synthesized and characterized, and their compatibility with rhBMP-2-induced osteoinduction was investigated. The results indicated that polymers with a controlled "solubility <--> insolubility" transition temperature could be prepared from N-isopropylacrylamide, ethylmethacrylate, and N-acryloxysuccinimide (NASI). NASI-containing polymers were able to conjugate to rhBMP-2 without additional cross-linkers. Implantation in the rat ectopic model, where alkaline phosphatase and calcium deposition were utilized as markers of osteoinductive activity, indicated that rhBMP-2 mixed with the polymers were effective for osteoinduction. Moreover, rhBMP-2 conjugated to the chosen polymers was as effective as native rhBMP-2 in inducing ALP activity and calcium deposition. We conclude that thermoreversible polymers are compatible with rhBMP-2-induced osteogenesis and can serve as novel biomaterials for rhBMP-2 delivery.

Neuroprotective effects of encapsulated CNTF-producing cells in a rodent model of Huntington's disease are dependent on the proximity of the implant to the lesioned striatum.

Huntington's disease (HD) is a devastating genetic disorder with no effective treatments for preventing or lessening the underlying neuronal degeneration. Intracerebral delivery of CNTF in animal models of HD has shown considerable promise as a means of protecting striatal neurons that would otherwise be destined to die. The present study examines whether the neuroprotective effects of CNTF require that the delivery be immediately proximal to the lesion site or whether protective effects can be exerted when the delivery site is more distal to the site of injury. Encapsulated CNTF-producing cells were implanted into the lateral ventricle either ipsilateral or contralateral to an intrastriatal quinolinic acid (QA) injection. A robust neuroprotective effect was observed only in those animals receiving CNTF implants ipsilateral to the QA injection. In these animals, the loss of striatal ChAT and GAD activity as well as the behavioral impairments that resulted from QA were completely prevented. In contrast, no neurochemical or behavioral benefits were produced by implants of CNTF-producing cells in the contralateral ventricle. These data continue to support the use of cellular delivery of CNTF for HD but caution that delivery directly to the striatum may be needed if any clinical benefits are to be seen.

Injection of chemotherapeutic microspheres and glioma. III: Parameters to optimize efficacy.

Injectable microspheres may provide a means of providing local, sustained exposure of glioma to chemotherapeutics to improve patient survival. Using a rodent model of surgically resected glioma, we previously demonstrated that direct injections of chemotherapeutic microspheres into the tissue surrounding a resection cavity provide superior survival effects over injections of the same microspheres directly into the surgical cavity. The present experiments extended this novel observation by exploring several parameters related to the use of intraparenchymal injections of chemotherapeutic microspheres to treat glioma. Using a rat model of resected glioma, several principles regarding the use of local sustained release carboplatin microspheres were established. First, an inverted U dose-response was observed, wherein further dose escalation beyond the optimal dose was not efficacious and indeed produced significant local toxicity. Second, it was necessary to expose approximately 40% of the tumor margin to sustained release carboplatin in order to increase survival in this model. Survival was not enhanced when the proportion of the tumor margin exposed to carboplatin was only 20%. Third, the distribution of the chemotherapeutic microsphere injections along the tumor perimeter was shown to be important, requiring that the entire perimeter be proportionately exposed to the chemotherapeutic agent. Together, these data continue to support the development of chemotherapeutic microspheres for treating glioma. However, they also caution that a number of fundamental parameters can profoundly influence the efficacy that might be expected from local sustained delivery. Careful attention to these principles is not only required if chemotherapeutic microspheres are to be used efficaciously, but these principles should provide a foundation to further optimize the potential of this and other polymeric delivery systems under development for local, intraparenchymal drug delivery to glioma.