Enhancement of drug delivery to bone: characterization of human tissue-nonspecific alkaline phosphatase tagged with an acidic oligopeptide

Hypophosphatasia is caused by deficiency of activity of the tissue-nonspecific alkaline phosphatase (TNSALP), resulting in a defect of bone mineralization. Enzyme replacement therapy (ERT) with partially purified plasma enzyme was attempted but with little clinical improvement. Attaining clinical effectiveness with ERT for hypophosphatasia may require delivering functional TNSALP enzyme to bone. We tagged the C-terminal-anchorless TNSALP enzyme with an acidic oligopeptide (a six or eight residue stretch of L-Asp), and compared the biochemical properties of the purified tagged and untagged enzymes derived from Chinese hamster ovary cell lines. The specific activities of the purified enzymes tagged with the acidic oligopeptide were the same as the untagged enzyme. In vitro affinity experiments showed the tagged enzymes had 30-fold higher affinity for hydroxyapatite than the untagged enzyme. Lectin affinity chromatography for carbohydrate structure showed little difference among the three enzymes. Biodistribution pattern from single infusion of the fluorescence-labeled enzymes into mice showed delayed clearance from the plasma up to 18 h post infusion and the amount of tagged enzyme retained in bone was 4-fold greater than that of the untagged enzyme. In vitro mineralization assays with the bone marrow from a hypophosphatasia patient using each of the three enzymes in the presence of high concentrations of pyrophosphate provided evidence of bone mineralization. These results show the anchorless enzymes tagged with an acidic oligopeptide are delivered efficiently to bone and function bioactively in bone mineralization, at least in vitro. They suggest potential advantages for use of these tagged enzymes in ERT for hypophosphatasia, which should be explored.     

Injection of oxotremorine in nucleus accumbens shell reduces cocaine but not food self-administration in rats

Mesencephalic dopamine neurons form synapses with acetylcholine (ACh)-containing interneurons in the nucleus accumbens (NAcc). Although their involvement in drug reward has not been systematically investigated, these large aspiny interneurons may serve an important integrative function. We previously found that repeated activation of nicotinic cholinergic receptors enhanced cocaine intake in rats but the role of muscarinic receptors in drug reward is less clear. Here we examined the impact of local changes in muscarinic receptor activation within the NAcc on cocaine and food self-administration in rats trained on a progressive ratio (PR) schedule of reinforcement. Animals were given a minimum of 9 continuous days of drug access before testing in order to establish a stable breaking point (BP) for intravenous cocaine infusions (0.75 mg/kg/infusion). Rats in the food group acquired stable responding on the PR schedule within 7 days. On the test day, rats were bilaterally infused in the NAcc with the muscarinic receptor agonist oxotremorine methiodide (OXO: 0.1, 0.3 or 1 nmol/side), OXO plus the M(1) selective antagonist pirenzepine (PIRENZ; 0.3 nmol/side) or aCSF 15 min before cocaine or food access. OXO dose dependently reduced BP values for cocaine reinforcement (-17%, -44% [p<0.05] and -91% [p<0.0001] for 0.1, 0.3 and 1.0 nmol, respectively) and these reductions dissipated by the following session. Pretreatment with PIRENZ blocked the BP-reducing effect of 0.3 nmol OXO. Notably, OXO (0.1, 0.3 and 1.0 nmol/side) injection in the NAcc did not affect BP for food reward. The results suggest that muscarinic ACh receptors in the caudomedial NAcc may play a role in mediating the behavior reinforcing effects of cocaine.     

Paramedics can apply and read Mantoux tuberculosis tests

Selected paramedics (26) received 2 h of training in the application and interpretation of Mantoux skin tests by a specially trained registered nurse. Instruction included a lecture, a video tape, and practice skin test administration on “induration arms.” All students successfully completed written and skills tests. During a follow-up period, registered nurses directly observed skin test application and interpretation by the paramedics when applying the skin test to other departmental fire fighters and compared these to set criteria. Interpretation of skin test results included appropriate referral for positive results. Paramedics administered 319 skin tests during subsequent follow-up and 305/319 (96%) were monitored by a registered nurse. All administration criteria were performed as indicated 100% of the time except for the criterion requiring a 5–10 mm wheal. The application was appropriately repeated in each case. In one application, a paramedic began to use the same needle and syringe to draw up additional solution, but was immediately corrected. Among 227 cases of test interpretation by paramedics, paramedics were monitored by a registered nurse in 169 interpretations (74%). All monitoring criteria were performed as indicated 100% of the time. Nurses concurred with each monitored measurement. Induration results were 0 mm, 216 tests; 1–9 mm, 9 tests; ≥10 mm, 2 tests. Appropriate follow-up was provided to the two individuals with ≥10 mm induration. The results indicate that paramedics can successfully apply and accurately interpret Mantoux skin tests on public safety employees, although the number of positive tests was low, reducing the reliability of the findings.     

Relationships among serum cystatin C,serum creatinine,lean tissue mass and glomerular filtration rate in healthy adults

In an effort to increase our knowledge of the optimal use of serum cystatin C and creatinine as glomerular filtration rate (GFR) markers, these variables, as well as lean tissue mass and GFR, were determined in a population of 42 healthy young adults (men and women with normal GFR). Dual­energy X­ray absorptiometry and measurement of the plasma clearance of iohexol were used to measure lean tissue mass and GFR, respectively. Serum creatinine was significantly correlated to lean tissue mass (r=0.65; p&lt;0.0001) but not to GFR (1/creatinine vs. GFR: r=0.11; p=0.106). In contrast, serum cystatin C correlated with GFR (1/cystatin C vs. GFR: r=0.32; p=0.0387), especially in men (1/cystatin C vs. GFR: r=0.64; p=0.0055), but not to lean tissue mass. These results might explain previous observations that serum cystatin C seems to be a better marker for GFR than serum creatinine, particularly for individuals with small to moderate decreases in GFR. However, the results also show that the serum concentrations of both creatinine and cystatin C are determined not only by GFR, but also by other factors. Since these additional factors differ for cystatin C and creatinine, it seems justified to use serum creatinine and cystatin C in conjunction to estimate GFR, at least until it is known in what situations serum creatinine or cystatin C is the preferable marker.     

Temporal changes of the plasma levels of cystatin C, β‐trace protein, β2‐microglobulin,urate and creatinine during pregnancy indicate continuous alterations in the renal filtration process

Objective. To determine the plasma levels of the renal functional markers creatinine, urate, cystatin C, β₂‐microglobulin and β‐trace protein in samples from the first, second, early third and late third trimesters of 398 healthy women with uncomplicated singleton pregnancies. Material and methods. Plasma samples from 58 healthy non‐pregnant women served as controls. The creatinine levels were significantly lower at all time‐points in pregnancy, whereas the urate levels were lower during the first and second trimesters but increased in the late third trimester. The cystatin C, β₂‐microglobulin and β‐trace protein levels displayed similar changes with increased levels in the third trimester but unaltered levels during the first and second trimesters. Results. The results indicate an increased filtration of low‐molecular weight molecules during pregnancy, particularly during the first and second trimesters, whereas filtration of 10–30?kDa molecules is decreased in the third but unaltered in the first and second trimesters. The levels of albumin and α₂‐macroglobulin were measured in the same samples. Conclusions. The albumin levels decreased in the second and third trimesters, whereas the levels of α₂‐macroglobulin were unchanged, which is compatible with a virtually unaltered transfer of α₂‐macroglobulin between the intra‐ and extravascular space during pregnancy and a significantly increased extravascular fraction of albumin.     

Acidic amino acid tag enhances response to enzyme replacement in mucopolysaccharidosis type VII mice

We have tested an acidic oligopeptide-based targeting system for delivery of enzymes to tissues, especially bone and brain, in a murine mucopolysaccharidosis type VII (MPS VII) model. This strategy is based upon tagging a short peptide consisting of acidic amino acids (AAA) to N terminus of human beta-glucuronidase (GUS). The pharmacokinetics, biodistribution, and the pathological effect on MPS VII mouse after 12 weekly infusions were determined for recombinant human untagged and tagged GUS. The tagged GUS was taken up by MPS VII fibroblasts in a mannose 6-phosphate receptor-dependent manner. Intravenously injected AAA-tagged enzyme had five times more prolonged blood clearance compared with the untagged enzyme. The tagged enzyme was delivered effectively to bone, bone marrow, and brain in MPS VII mice and was effective in reversing the storage pathology. The storage in osteoblasts was cleared similarly with both enzyme types. However, cartilage showed a little response to any of the enzymes. The tagged enzyme reduced storage in cortical neurons, hippocampus, and glia cells. A highly sensitive method of tandem mass spectrometry on serum indicated that the concentration of serum dermatan sulfate and heparan sulfate in mice treated with the tagged enzyme decreased more than the untagged enzyme. These preclinical studies suggest that this AAA-based targeting system may enhance enzyme-replacement therapy.