Intravenous tolerability of an acid vehicle in Sprague Dawley rats and beagle dogs after daily slow bolus injection and infusion for one hour during 14 days

To assess the tolerability of an acid vehicle to be used in toxicology studies, a low pH aqueous solution containing 16.4 mg/ml of citric acid, 4.2 mg/ml of disodium phosphate, 25 mg/ml of mannitol, adjusted with phosphoric acid/NaOH 1 M to pH 3 was daily administered intravenously to rats and dogs for 14 consecutive days. The dosing regimen consisted of a slow intravenous bolus injection given over 30 s (0.75 and 0.625 ml/kg, for rats and dogs, respectively) followed by intravenous infusion for one hour (3.75 and 2.75 ml/kg/h, for rats and dogs, respectively). In rats, the dose was administered via the lateral tail vein. In dogs, the intravenous bolus dose was administered via the vena cephalica, vena saphena or vena jugularis, whilst the infusion dose was given into the vena cephalica or vena saphena. In rats, administration of the vehicle was associated with clinical signs (occasional mild vocalization and agitation) which were considered to be due to local irritation during the dosing procedure. Nevertheless, only mild histopathological changes at the injection site were found, while no relevant clinical chemistry changes were found in this species. However, the vehicle caused significant vascular damage with thrombus formation in the dog. It is therefore concluded that this vehicle is suitable for 2-week rat toxicity studies, if carefully applied. The vehicle with its present regimen should not be used in dogs, in view of the prohibitive findings.     

Radiosensitization by gemcitabine fixed-dose-rate infusion versus bolus injection in a pancreatic cancer model

It has recently been shown that fixed-dose-rate (gemcitabine) infusion may be superior to bolus gemcitabine in the treatment of metastatic pancreas cancer. We wished to compare the radiosensitizing effects of fixed-dose-rate gemcitabine infusion to standard bolus injection. We measured weight loss and mouse intestinal crypt survival to determine equally toxic concentrations of gemcitabine administered through a 3-hour fixed-dose-rate infusion versus bolus injection in combination with fractionated radiation. To measure the effect of fixed-dose-rate gemcitabine infusion or bolus injection on radiosensitization, we treated mice bearing Panc-1 xenografts with equally toxic concentrations of gemcitabine (100 mg/kg fixed-dose-rate infusion or 500 mg/kg bolus injection) and fractionated radiation and monitored tumor growth. We found that 100 mg/kg gemcitabine through fixed-dose-rate infusion produced the same weight loss and intestinal crypt toxicity as the 500 mg/kg bolus injection. In nude mice bearing Panc-1 xenografts, fixed-dose-rate gemcitabine infusion produced greater radiosensitization than bolus injection with tumor doubling times of 44 +/- 5 versus 29 +/- 3 days, respectively (*P < .05). Fixed-dose-rate gemcitabine infusion produced enhanced radiosensitization without additional normal tissue toxicity compared to bolus gemcitabine injection. These data support an ongoing clinical trial using fixed-dose-rate gemcitabine infusion combined with conformal radiation in the treatment of locally advanced pancreatic cancer.     

Acute toxicology and pharmacokinetic assessment of a ribozyme (ANGIOZYME) targeting vascular endothelial growth factor receptor mRNA in the cynomolgus monkey

The potential acute toxicity of a ribozyme (ANGIOZYME) targeting the flt-1 vascular endothelial growth factor (VEGF) receptor mRNA was evaluated in cynomolgus monkeys following i.v. infusion or s.c. injection. ANGIOZYME was administered as a 4-hour i.v. infusion at doses of 10, 30, or 100 mg/kg or a s.c. bolus at 100 mg/kg. End points included blood pressure, electrocardiogram (ECG), clinical chemistry, hematology, complement factors, coagulation parameters, and ribozyme plasma concentrations. ANGIOZYME was well tolerated, with no drug-associated morbidity or mortality. There was no clear evidence of ANGIOZYME-related adverse effects in this study. Slight increases in spleen weight and lymphoid hyperplasia were observed in several animals. However, these changes were not dose dependent. Steady-state concentrations of ANGIOZYME were achieved during the 4-hour infusion of 10, 30, or 100 mg/kg. Dose-dependent elimination of ANGIOZYME was observed, with faster clearance at the two highest doses. ANGIOZYME was slowly absorbed after s.c. administration, resulting in steady-state concentrations for the 9-hour sampling period. Monkeys in this toxicology study received significant plasma ANGIOZYME exposure by both the s.c. and i.v. routes.     

Targeted tumor therapy with the TGF-beta 2 antisense compound AP 12009

TGF-beta overexpression is a hallmark of various malignant tumors. This is due to the pivotal role of TGF-beta as it regulates key mechanisms of tumor development, namely immunosuppression, metastasis, angiogenesis, and proliferation. We have developed a new immunotherapeutic approach for the treatment of malignant tumors based on the specific inhibition of TGF-beta2 by the antisense oligodeoxynucleotide AP 12009. After providing preclinical proof of concept, we assessed safety and efficacy of AP 12009 in clinical phase I/II open-label dose escalation studies in high-grade glioma patients. Median survival time after recurrence exceeded the up to date literature data for chemotherapy. A phase I/II study in pancreatic carcinoma and malignant melanoma is currently ongoing. Our results implicate targeted TGF-beta2 suppression as a promising therapeutic approach for malignant tumor therapy.     

Assessment of hydroxypropyl methylcellulose, propylene glycol, polysorbate 80, and hydroxypropyl-β-cyclodextrin for use in developmental and reproductive toxicology studies

BACKGROUND: A series of studies were conducted to assess Polysorbate 80 (PS80), Propylene Glycol (PG), and Hydroxypropyl‐β‐Cyclodextrin (HPβCD), when compared with Hydroxypropyl Methylcellulose (MC) in developmental and reproductive toxicology (DART) studies. METHODS: In the rat fertility study, 20 mg/kg MC, 10 mg/kg PS80, 1,000 mg/kg PG, 500 mg/kg HPβCD or 1,000 mg/kg HPβCD were administered orally before/during mating, and on gestation Day (GD) 0–7, followed by an assessment of embryonic development on GD 14. In the rat and rabbit teratology studies, the doses of MC, PS80, PG, and HPβCD were the same as those in the fertility study. In these teratology studies, pregnant females were dosed during the period of organogenesis, followed by an assessment of fetal external, visceral, and skeletal development. RESULTS: In the rat fertility and rat teratology studies, PS80, PG, and HPβCD did not exhibit toxicity, when compared with MC. Similarly, in the rabbit teratology study, there was no PS80 or PG‐related toxicity, when compared with MC. However, individual rabbits in the 500 and 1,000 mg/kg HPβCD groups exhibited maternal toxicity, which included stool findings, decreased food consumption, and body weight gain. Furthermore, one rabbit each in the 500 and 1,000 mg/kg HPβCD groups exhibited evidence of abortion, which was considered secondary to maternal toxicity. CONCLUSIONS: Although HPβCD was not well tolerated in rabbits at doses of 500 and 1,000 mg/kg, PS80 and PG were comparable to MC and should be considered for use in developmental and reproductive toxicology studies. Birth Defects Res (Part B) 89:504–516,2010. © 2010 Wiley‐Liss, Inc.     

Continuous intravenous infusion and simultaneous pharmacokinetic monitoring in ambulatory rabbits with a programmable micropump and dual subcutaneous Silastic central venous catheters.

We describe a newly developed method of continuous intravenous infusion and simultaneous monitoring of plasma levels of investigational compounds in ambulatory untethered rabbits. Continuous infusion was administered by means of a portable programmable external micropump which permitted adjustable dosing. Simultaneous plasma pharmacokinetic monitoring during infusion was accomplished by dual silastic central venous catheters. The potential applications of the micropump infusion system as an alternative to current methods of continuous infusion in other species and in other studies are further discussed. This method provided a safe, reliable, and well-tolerated method of adjustable continuous intravenous infusion and simultaneous sampling of central venous blood in rabbits.     

Left ventricular, systemic arterial, and baroreflex responses to ketamine and TEE in chronically instrumented monkeys

Effects of prescribed doses of ketamine five minutes after application and influences of transesophageal echocardiography (TEE) on left ventricular, systemic arterial, and baroreflex responses were investigated to test the hypothesis that ketamine and/or TEE probe insertion alter cardiovascular function. Seven rhesus monkeys were tested under each of four randomly selected experimental conditions: (1) intravenous bolus dose of ketamine (0.5 ml), (2) continuous infusion of ketamine (500 mg/kg/min), (3) continuous infusion of ketamine (500 mg/kg/min) with TEE, and (4) control (no ketamine or TEE). Monkeys were chronically instrumented with a high fidelity, dual-sensor micromanometer to measure left ventricular and aortic pressure and a transit-time ultrasound probe to measure aortic flow. These measures were used to calculate left ventricular function. A 4-element Windkessel lumped-parameter model was used to estimate total peripheral resistance and systemic arterial compliance. Baroreflex response was calculated as the change in R-R interval divided by the change in mean aortic pressure measured during administration of graded concentrations of nitroprusside. The results indicated that five minutes after ketamine application heart rate and left ventricular diastolic compliance decreased while TEE increased aortic systolic and diastolic pressure. We conclude that ketamine may be administered as either a bolus or continuous infusion without affecting cardiovascular function 5 minutes after application while the insertion of a TEE probe will increase aortic pressure. The results for both ketamine and TEE illustrate the classic "Hawthorne Effect," where the observed values are partly a function of the measurement process. Measures of aortic pressure, heart rate, and left ventricular diastolic pressure should be viewed as relative, as opposed to absolute, when organisms are sedated with ketamine or instrumented with a TEE probe.     

Non-invasive Monitoring of L-2-Oxothiazolidine-4-Carboxylate Metabolism in the Rat Brain by In vivo 13C Magnetic Resonance Spectroscopy

The cysteine precursor L-2-oxothiazolidine-4-carboxylate (OTZ, procysteine) can raise cysteine concentration, and thus glutathione levels, in some tissues. OTZ has therefore been proposed as a prodrug for combating oxidative stress. We have synthesized stable isotope labeled OTZ (i.e. L-2-oxo-[5-(13)C]-thiazolidine-4-carboxylate, (13)C-OTZ) and tracked its uptake and metabolism in vivo in rat brain by (13)C magnetic resonance spectroscopy. Although uptake and clearance of (13)C-OTZ was detectable in rat brain following a bolus dose by in vivo spectroscopy, no incorporation of isotope label into brain glutathione was detectable. Continuous infusion of (13)C-OTZ over 20 h, however, resulted in (13)C-label incorporation into glutathione, taurine, hypotaurine and lactate at levels sufficient for detection by in vivo magnetic resonance spectroscopy. Examination of brain tissue extracts by mass spectrometry confirmed only low levels of isotope incorporation into glutathione in rats treated with a bolus dose and much higher levels after 20 h of continuous infusion. In contrast to some previous studies, bolus administration of OTZ did not alter brain glutathione levels. Even a continuous infusion of OTZ over 20 h failed to raise brain glutathione levels. These studies demonstrate the utility of in vivo magnetic resonance for non-invasive monitoring of antioxidant uptake and metabolism in intact brain. These types of experiments can be used to evaluate the efficacy of various interventions for maintenance of brain glutathione.     

Influence of rostral ventrolateral medulla on renal sympathetic baroreflex in conscious rabbits

Previous studies with anesthetized animals have shown that the pressor region of the rostral ventrolateral medulla (RVLM) is a critical site in vasomotor control. The aim of this study was to develop, in conscious rabbits, a technique for microinjecting into the RVLM and to determine the influence of this area on renal sympathetic nerve activity (RSNA) and arterial pressure (AP) using local injections of glutamate, rilmenidine, ANG II and sarile. Rabbits were implanted with guide cannulas for bilateral microinjections into the RVLM (n = 7) or into the intermediate ventrolateral medulla (IVLM, n = 6) and an electrode for measuring RSNA. After 7 days of recovery, injections of glutamate (10 and 20 nmol) into the RVLM increased RSNA by 81 and 88% and AP by 17 and 25 mmHg, respectively. Infusion of glutamate (2 nmol/min) into the RVLM increased AP by 15 mmHg and the RSNA baroreflex range by 38%. By contrast, injection of the imidazoline receptor agonist rilmenidine (4 nmol) into the RVLM decreased AP by 8 mmHg and the RSNA baroreflex range by 37%. Injections of rilmenidine into the IVLM did not alter AP or RSNA. Surprisingly, treatments with ANG II (4 pmol/min) or the ANG II receptor antagonist sarile (500 pmol) into the RVLM did not affect the resting or baroreflex parameters. Infusion of ANG II (4 pmol/min) into the fourth ventricle increased AP and facilitated the RSNA baroreflex. Our results show that agents administered via a novel microinjecting system for conscious rabbits can selectively modulate neuronal activity in circumscribed regions of the ventrolateral medulla. We conclude that the RVLM plays a key role in circulatory control in conscious rabbits. However, we find no evidence for the role of ANG II receptors in the RVLM in the moment-to-moment regulation of AP and RSNA.     

Inhibition of TGF-beta2 with AP 12009 in recurrent malignant gliomas: from preclinical to phase I/II studies

Transforming growth factor-beta2 (TGF-beta2) is known to suppress the immune response to cancer cells and plays a pivotal role in tumor progression by regulating key mechanisms including proliferation, metastasis, and angiogenesis. For targeted protein suppression the TGF-beta2-specific antisense oligodeoxynucleotide AP 12009 was developed. In vitro experiments have been performed to prove specificity and efficacy of the TGF-beta2 inhibitor AP 12009 employing patient-derived malignant glioma cells as well as peripheral blood mononuclear cells (PBMCs) from patients. Clinically, the antisense compound AP 12009 was assessed in three Phase I/II-studies for the treatment of patients with recurrent or refractory malignant (high-grade) glioma WHO grade III or IV. Although the study was not primarily designed as an efficacy evaluation, prolonged survival compared to literature data and response data were observed, which are very rarely seen in this tumor indication. Two patients experienced long-lasting complete tumor remissions. These results implicate targeted TGF-beta2-suppression using AP 12009 as a promising novel approach for malignant gliomas and other highly aggressive, TGF-beta-2-overexpressing tumors.     

Preclinical evaluation of anti-CD86 immunotoxin in rhesus monkeys: analysis of systemic toxicity,pharmacokinetics, and effect on primary T-cell responses

CD86 and CD80 costimulatory antigens are highly overexpressed on Hodgkin/Reed-Sternberg cells in patients with Hodgkin's disease (HD) and are candidate target antigens for immunotoxins in order to eliminate minimal residual disease. In this study we have evaluated the pharmacokinetics (and immunological) toxicity in rhesus monkeys of immunotoxins consisting of gelonin conjugated to anti-CD86 (alphaCD86-IT). Both alphaCD86-IT and alphaCD80-IT inhibited protein synthesis in B cell lines from rhesus monkeys and inhibited the mixed leukocyte reaction. Reactivity of the alphaCD86 antibody with rhesus monkey CD86 (RhCD86) was shown by cloning and transfecting RhCD86, which conferred reactivity of the alphaCD86 antibody used in this study. alphaCD86-IT was administered as single intravenous bolus injection in four rhesus monkeys, and achieved plasma concentration in 50-fold excess able to eliminate cultured Hodgkin/Reed-Sternberg cells up to 6 h. The animals were capable of generating primary immune responses to both gelonin and murine IgG within 9 days after infusion with alphaCD86-IT. No evidence could be found of significant (immunological) toxicity. The results of this study show that alphaCD86-IT can be applied safely in an effective dose.     

The relationship between free and total calcium concentrations in the matrix of liver and brain mitochondria

Three sequential phases of mitochondrial calcium accumulation can be distinguished: matrix dehydrogenase regulation, buffering of extramitochondrial free calcium, and finally activation of the permeability transition. Relationships between these phases, free and total matrix calcium concentration, and phosphate concentration are investigated in rat liver and brain mitochondria. Slow, continuous calcium infusion is employed to avoid transient bioenergetic consequences of bolus additions. Liver and brain mitochondria undergo permeability transitions at precise matrix calcium loads that are independent of infusion rate. Cytochrome c release precedes the permeability transition. Cyclosporin A enhances the loading capacity in the presence or absence of acetoacetate. A remarkably constant free matrix calcium concentration, in the range 1-5 microM as monitored by matrix-loaded fura2-FF, was observed when total matrix calcium was increased from 10 to at least 500 nmol of calcium/mg of protein. Increasing phosphate decreased both the free matrix calcium and the matrix calcium-loading capacity. Thus the permeability transition is not triggered by a critical matrix free calcium concentration. The rate of hydrogen peroxide detection by Amplex Red decreased during calcium infusion arguing against a role for oxidative stress in permeability pore activation in this model. A transition between a variable and buffered matrix free calcium concentration occurred at 10 nmol of total matrix calcium/mg protein. The solubility product of amorphous Ca3(PO4)2 is consistent with the observed matrix free calcium concentration, and the matrix pH is proposed to play the major role in maintaining the low matrix free calcium concentration.     

Influence of D2-receptor blockade on the nitrergic system activity of the nucleus accumbens

In Sprague-Dawley rats, by means of in vivo microdialysis combined with HPLC analysis it was shown that the infusion into the n. accumbens of the D2-receptor antagonist raclopride (20, 100 microM) did not affect extracellular level ofcitrulline (an NO co-product) in this brain area. The intraaccumbal infusion of NMDA, an NMDA receptor agonist (100 microM) caused a rise of the extracellular citrulline level in this brain area. This rise was prevented by intraaccumbal infusion of 0.5 mM 7-nitroindazole, a neuronal NO-synthase inhibitor, and it was significantly reduced by the infusion of raclopride (20, 100 microM) into this brain area. The data obtained suggest that the D2-receptors of the n. accumbens are implicated in the regulation of neuronal NO-synthase activity induced by local NMDA receptor stimulation.     

Ethyl t‐butyl ether: review of reproductive and developmental toxicity

Ethyl t‐butyl ether (ETBE) is a motor fuel oxygenate used in reformulated gasoline. Knowledge of developmental and reproductive toxicity potential of ETBE is critical for making informed decisions about acceptance and regulations. This review discusses toxicology studies providing information about effects on reproduction and the conceptus. Seven GLP‐compliant studies following widely accepted protocols have focused specifically on developmental and reproductive toxicity (DART) in rats and rabbits exposed to ETBE by gavage with doses up to 1,000 mg/kg body weight/day, the limit specified in standardized test guidelines. Other repeat‐dose general toxicology studies have administered ETBE to rodents for up to 180 days, and included reproductive organ weights, histology, or other indications of reproductive system structure or function. DART potential of the main ETBE metabolite t‐butyl alcohol and class‐related MTBE has also been studied. More GLP‐compliant studies exist for evaluating ETBE using well‐established, currently recommended protocols than are available for many other chemicals used today. The database for determining ETBE DART potential is adequate, although not all study details are currently easily accessible for peer‐review. ETBE does not appear to be selectively toxic to reproduction or embryofetal development in the absence of other manifestations of general toxicity. Studies using recommended methods for sample preservation and analysis have shown no targeted effect on the reproductive system. No embryofetal effects were observed in rabbits. Early postnatal rat pup deaths show no clear dose‐response and have largely been attributed to total litter losses with accompanying evidence of maternal neglect or frank maternal morbidity. Birth Defects Res (Part B) 89:239–263, 2010. © 2010 Wiley‐Liss, Inc.     

Embryotoxicity studies of norfloxacin in cynomolgus monkeys: I. Teratology studies and norfloxacin plasma concentration in pregnant and nonpregnant monkeys

Norfloxacin, a new orally active antibiotic, was investigated in cynomolgus monkeys for potential developmental toxicity. Fifty-seven monkeys were administered a control vehicle or norfloxacin by nasogastric gavage during the major period of organogenesis on gestational days (GD) 21 through 50 at doses of 0, 50, 100, 150, or 200/300 mg/kg/day. There was no evidence of teratogenicity at any dose level. Maternotoxicity and a significant increase in embryolethality occurred following doses of 200/300 mg/kg/day. The maternotoxicity was not expected based on range-finding studies in nonpregnant female monkeys, which showed no signs of toxicity in doses up to 500 mg/kg/day. Additional studies were conducted to determine if norfloxacin caused similar toxicity later in gestation. Forty-six pregnant monkeys were dosed with a control vehicle or 200 mg/kg/day norfloxacin for one of three 10-day periods on GD 36-45, 71-80, or 111-120. There were no maternotoxic, embryotoxic, or fetotoxic effects observed. Plasma concentrations of norfloxacin in five cynomolgus monkeys following 50 and 200 mg/kg oral doses were not dose-proportionate. However, at a given dose, administered in cross-over fashion, plasma concentrations of norfloxacin were higher in nonpregnant females (approximately 20-40%) than during pregnancy when the same subject was compared. At the no-observed-effect dose for maternal and embryotoxicity (50 mg/kg), peak plasma concentrations of norfloxacin in pregnant cynomolgus monkeys are approximately threefold higher than those observed in human volunteers receiving norfloxacin at the maximum recommended therapeutic dose of 400 mg (5.7 mg/kg based on 70 kg body weight) twice per day.     

Uptake and Metabolism of Choline by Rat Brain After Acute Choline Administration

The present study is concerned with the uptake and metabolism of choline by the rat brain. Intraperitoneal administration of choline chloride (4-60 mg/kg) caused a dose-dependent elevation of the plasma choline concentration from 11.8 to up to 165.2 microM within 10 min and the reversal of the negative arteriovenous difference (AVD) of choline across the brain to positive values at plasma choline levels of greater than 23 microM. Net choline release and uptake were linearly dependent on the plasma choline level in the physiological range of 10-50 microM, whereas the CSF choline level was significantly increased only at plasma choline levels of greater than 50 microM. The bolus injection of 60 mg/kg of [3H]choline chloride caused the net uptake of greater than 500 nmol/g of choline by the brain as calculated from the AVD, which was reflected in a minor increase of free choline level and a long-lasting increase of brain phosphorylcholine content, which paralleled the uptake curve. Loss of label from phosphorylcholine 30 min to 24 h after choline administration was accompanied by an increase of label in phosphatidylcholine, an indication of a delayed transfer of newly taken-up choline into membrane choline pools. In conclusion, homeostasis of brain choline is maintained by a complex system that interrelates choline net movements into and out of the brain and choline incorporation into and release from phospholipids.     

Comparison of the developmental toxicity of aspirin in rabbits when administered throughout organogenesis or during sensitive windows of development

BACKGROUND: A review of the nonsteroidal anti‐inflammatory drug (NSAID) literature suggested occurrences of low‐level incidences of cardiovascular and midline defects in rabbit fetuses exposed in utero. Aspirin (acetylsalicylic acid, ASA) is a widely used NSAID that irreversibly inhibits cyclooxygenases (COXs) 1 and 2. ASA has been studied extensively in rats and has consistently increased low‐incidence cardiovascular malformations and defects in midline closure. The objectives of the current study were to comprehensively define the developmental toxicology profile of ASA in rabbits by using a dosing paradigm encompassing the period of organogenesis and to test the hypothesis that maternal gastrointestinal toxicity after repeated dose administrations hampers the detection of low‐incidence malformations with ASA in rabbits by limiting ASA administration to sensitive windows for cardiovascular development and midline closure. METHODS: ASA was administered to pregnant New Zealand White rabbits from gestation days (GDs) 7 to 19 at dose levels of 125, 250, and 350 mg/kg per day and as single doses of 500, 750, or 1000 mg/kg on GD 9, 10, or 11. Cesarean sections were performed on GD 29, and the fetuses were examined for external, visceral, and skeletal development. RESULTS: In the repeated dose study, maternal toxicity was exhibited in the 250‐ and 350‐mg/kg per day groups by mortality and decreased food consumption and body weight gain. In the single dose studies, maternal toxicity was exhibited at all doses by reductions in body weight gain and food consumption for 3 days after treatment. Fetal body weight was significantly reduced in the repeated dose study at 350 mg/kg per day. Fetal weights were not affected by single doses of ASA on GD 9, 10, or 11. There were no treatment‐related external, visceral, or skeletal malformations associated with ASA administration throughout organogenesis or with single doses administered during critical developmental windows. CONCLUSION: These findings supported previous work demonstrating that ASA is not teratogenic in rabbits, as opposed to rats, even when large doses are administered on single days during specific windows of development. Birth Defects Research (Part B) 68:38–46, 2003. © 2003 Wiley‐Liss, Inc.     

Taurine kinetics assessed using [1,2-13C2]taurine in healthy adult humans

To assess the dynamics of taurine metabolism in vivo, two sets of studies were carried out in healthy volunteers. First, pilot studies were carried in a single human subject to determine the time course of plasma and whole blood isotope enrichment over the course of an 8-h, unprimed continuous infusion of [1,2-(13)C(2)]taurine. Second, five healthy adult males received two tracer infusions on separate days and in randomized order: 1) a 6-h continuous infusion of [1,2-(13)C(2)]taurine (3.1 +/- 0.2 micromol x kg(-1) x h(-1)) and 2) a bolus injection of [(13)C(2)]taurine (3.0 +/- 0.1 micromol/kg). Isotope enrichments in plasma and whole blood taurine were determined by gas chromatography-mass spectrometry. The pilot experiments allowed us to establish that steady-state isotope enrichment was reached in plasma and whole blood by the 5th h of tracer infusion. The plateau enrichment reached in whole blood was lower than that obtained in plasma taurine (P < 0.02). In the second set of studies, the appearance rate (R(a)) of plasma taurine, determined from continuous infusion studies was 31.8 +/- 3.1 micromol x kg(-1) x h(-1). After a bolus injection of tracer, the enrichment decay over the subsequent 2 h was best fitted by a two-exponential curve. Taurine R(a) was approximately 85% higher when determined using the bolus injection technique compared with continuous infusion of tracer. We conclude that 1) taurine R(a) into plasma is very low in healthy postabsorptive humans, and, due to taurine compartmentation between the extra- and intracellular milieus, may represent only interorgan taurine transfer and merely a small fraction of whole body taurine turnover; and 2) the bolus injection technique may overestimate taurine appearance into plasma. Further studies are warranted to determine whether alterations in bile taurine dynamics affect taurine R(a).     

Pre-clinical studies of retrovirally transduced human chondrocytes expressing transforming growth factor-beta-1 (TG-C)

Background aimsThe aim was to evaluate cartilage regeneration in animal models involving induced knee joint damage. Through cell-mediated gene therapy methods, a cell mixture comprising a 3:1 ratio of genetically unmodified human chondrocytes and transforming growth factor beta-1 (TGF-β1)-secreting human chondrocytes (TG-C), generated via retroviral transduction, resulted in successful cartilage proliferation in damaged regions.MethodsNon-clinical toxicology assessments for efficacy, biodistribution and local/systemic toxicity of single intra-articular administration of the cell mixture in mice, rabbits and goats was conducted.ResultsAdministration of the mixture was tolerated well in all of the species. There was evidence of cartilage proliferation in rabbits and goats. As an additional precautionary step, the efficacy of TGF-β1 secretion in irradiated human chondrocytes was also demonstrated.ConclusionsFour studies in rabbits and goats demonstrated the safety and efficacy of TG-C following direct intra-articular administration in animal models involving induced knee joint damage. Based on these pre-clinical studies authorization has been received from the USA Food and Drug Administration (FDA) to proceed with an initial phase I clinical study of TG-C for degenerative arthritis.