Chronic neuropathic pain is accompanied by global changes in gene expression and shares pathobiology with neurodegenerative diseases.

Neuropathic pain is induced by injury or disease of the nervous system. Studies aimed at understanding the molecular pathophysiology of neuropathic pain have so far focused on a few known molecules and signaling pathways in neurons. However, the pathophysiology of neuropathic pain appears to be very complex and remains poorly understood. A global understanding of the molecular mechanisms involved in neuropathic pain is needed for a better understanding of the pathophysiology and treatment of neuropathic pain. Towards this end, we examined global gene expression changes as well as the pathobiology at the cellular level in a spinal nerve ligation neuropathic pain model using DNA microarray, quantitative real-time PCR and immunohistochemistry. We found that the behavioral hypersensitivity that is manifested in the persistent pain state is accompanied by previously undescribed changes in gene expression. In the DRG, we found regulation of: (1) immediate early genes; (2) genes such as ion channels and signaling molecules that contribute to the excitability of neurons; and (3) genes that are indicative of secondary events such as neuroinflammation. In addition, we studied gene regulation in both injured and uninjured DRG by quantitative PCR, and observed differential gene regulation in these two populations of DRGs. Furthermore, we demonstrated unexpected co-regulation of many genes, especially the activation of neuroinflammation markers in both the PNS and CNS. The results of our study provide a new picture of the molecular mechanisms that underlie the complexity of neuropathic pain and suggest that chronic pain shares common pathobiology with progressive neurodegenerative disease.

An extended point-area deconvolution approach for assessing drug input rates.

PURPOSE: To describe an extended point-area deconvolution approach for evaluating drug input rates based on the application of piecewise cubic polynomial functions. METHODS: Both the nonimpulse response data and the impulse reference data were independently represented by the piecewise cubic polynomials to obtain interpolations, numerical integration, and reduced step size for the staircase input rates. A moving average algorithm was employed to compute the input rate estimates. The method was illustrated using data from preclinical and human studies. Simulations were used to examine the effects of data noise. RESULTS: In all cases examined, the piecewise cubic interpolation functions combined with the moving average algorithm yielded estimates that were reasonable and acceptable. Compared to the standard point-area approach based on the trapezoidal rule, the present method resulted in estimates that were closer to the expected values. CONCLUSIONS: The point-area deconvolution analysis is one of the preferred approaches in assessing pharmacokinetic and biopharmaceutic data when it is undesirable to assume the functional forms of the input processes. The present method provides improved performance and greater flexibility of this approach.

Comments on Nedelman and Jia's extension of Satterthwaite's approximation applied to pharmacokinetics.

In this letter, I have pointed out how the estimator given by Nedelman and Jia can be made unbiased with only a small modification. Moreover, I report on a new simulation study to show that the unbiased estimator of psi has favorable statistical properties in terms of confidence interval width and coverage probability. Further, the simulation study shows the effect of enforcement of the C-S inequality on the performance of both the biased and unbiased estimators.

Effects of L-749,329, an ET(A)/ET(B) endothelin receptor antagonist, in a porcine coronary artery injury model of vascular restenosis.

BACKGROUND: Previous studies in animal models of angioplasty have suggested a role in neointimal hyperplasia for endothelins (ETs), potent vasoconstricting peptides that also exert growth-promoting effects. The present studies were undertaken to test the hypothesis that endothelin receptor blockade can reduce neointimal thickening in injured porcine coronary arteries. METHODS AND RESULTS: An ET(A)/ET(B) antagonist, L-749,329, was evaluated as an inhibitor of intimal thickening in a porcine balloon/stent model of coronary artery injury. L-749,329 competitively inhibited [(125)I]ET-1 binding to porcine ET(A) (IC(50) approximately 0.3 nmol/L) or ET(B) (IC(50) approximately 20 nmol/L) receptors and inhibited ET-1-stimulated signaling in cell culture. In anesthetized pigs, big ET-1-stimulated increases in systemic blood pressure were totally inhibited after intravenous infusion of L-749,329 (>/=0.2 mg. kg(-1). h(-1)). In vascular injury studies, pigs were treated with vehicle or L-749,329 (1 mg. kg(-1). h(-1)) beginning 2 days before and continuing 28 days after experimental angioplasty. Left anterior descending, left circumflex, and/or right coronary arteries were injured by inflation of an angioplasty balloon wrapped with a coiled metallic stent. After 28 days, mean neointimal thickness in the L-749,329-treated group was reduced by 9.0% compared with vehicle-treated controls, but this effect was not statistically significant (P=0.13). CONCLUSIONS: Blockade of endothelin receptors for 28 days with only a mixed ET(A)/ET(B) receptor antagonist is insufficient to substantially inhibit intimal hyperplasia after balloon/stent coronary artery injury in the pig, in contrast to results with a selective ET(A) antagonist. The effects of selective or mixed ET(A)/ET(B) antagonists in diseased vessels remain to be determined in this model.

Antigen levels and antibody titers after DNA vaccination.

DNA vaccination generates strong cellular and humoral immunity in animal models. The mechanisms by which plasmid DNA uptake and expression after intramuscular injection lead to immune responses are not well understood. In particular, the importance of antigen expression levels on subsequent antibody immune responses has not been established. We found that a chemiluminescent assay for alkaline phosphatase allows measurement of antigen levels of secreted alkaline phosphatase (SEAP) in vivo after intramuscular injection of a wide range of plasmid doses. The mice produced antibodies to the alkaline phosphatase reporter gene and both antigen levels and antibody titers were measured over time. We found that the correlation between initial antigen level and antibody response was high (r = 0.74, p < 0.001) and remained high even after accounting for the dose of plasmid injected (r = 0.61, p < 0.001). The correlation between DNA dose and antibody titer was statistically significant (r = 0.53, p < 0.001) but was reduced to almost zero after we accounted for initial antigen levels.

A small sample confidence interval approach to assess individual bioequivalence.

The concept of interchangeable pharmaceutical products has been examined in great detail in the literature. Anderson and Hauck proposed a statistical random coefficient model to study 'switchability', and coined the phrase 'individual bioequivalence' which they defined with a probability-based inequality. Since that paper there has been considerable work and discussion. The Food and Drug Administration has recommended the introduction of individual bioequivalence (IBE) and population bioequivalence (PBE) methods in a draft guidance document. The proposal in the draft guidance includes criteria for IBE and PBE and recommends the use of non-parametric bootstrap 95 per cent upper confidence intervals for the conclusion of either IBE or PBE. However, this method requires intensive computations. We have developed an alternative confidence interval procedure to assess IBE by the FDA recommended criteria. This method utilizes Howe's approximation I to a Cornish-Fisher expansion. Our proposed method is applicable to balanced or unbalanced data in a broad class of extended cross-over designs, and can be easily programmed using readily available software.

A method for estimating and testing area under the curve in serial sacrifice, batch, and complete data designs.

The extent of drug availability is often measured by the area under the concentration-time curve. In animal studies, experimental constraints can limit the number of observations available on each animal. Estimation of area under the curve and its standard error are straightforward when each animal is measured at each time point. Bailer and Nedelman et al., have described techniques for estimating the area under the curve and its standard error when each animal is measured once. Yeh has described a technique for the hybrid case where animals are measured more than once, but not at all time points. We describe a method for estimating area under the curve and its standard error which is applicable to all three types of designs. We give formulas for testing treatment differences, including dose trends and dose proportionality, in area under the curve for designs containing an arbitrary number of treatments. A jackknife estimator is also described.

Mapping of hKCa3 to chromosome 1q21 and investigation of linkage of CAG repeat polymorphism to schizophrenia.

CAG trinucleotide polymorphisms in the neuronal small conductance calcium-activated potassium channel gene hKCa3 have been reported to be associated with schizophrenia. Attempts to confirm this finding have met with mixed results. We investigated hKCa3 CAG allele lengths in families from the National Institute of Mental Health (NIMH) Schizophrenia Genetics Initiative, by comparing transmission to discordant siblings and parental transmission to affected offspring. Overall, there was no convincing evidence that hKCa3 CAG lengths differ between schizophrenics and controls. We did, however, observe a trend (P = 0.063) toward over-representation of long (> or = 19) CAG repeats in the shorter of the two hKCa3 alleles in schizophrenics. There was no evidence of excessive parental transmission of long CAG repeat alleles to affected offspring. In addition, we re-mapped hKCa3 and found that it resides on chromosome 1q21, in a region which has been linked to familial hemiplegic migraine, but not to schizophrenia. These data provide no significant support for the association of hKCa3 with schizophrenia.

Quantitation of mixed-base populations of HIV-1 variants by automated DNA sequencing with BODIPY dye-labeled primers.

Direct DNA sequencing of human immunodeficiency virus type 1 (HIV-1) pol and env gene regions was characterized for accuracy and precision. Restricted maximum likelihood (REML) analysis of molecular clone reconstruction experiments using a primer-walking strategy showed that the BODIPY and BODIPY energy-transfer (BET) dye primers sets were significantly more accurate in quantitating heterogenous base mixtures than the fluorescein/rhodamine dye primers. Of the three sets examined, the BET dye primers were the most accurate. The improved accuracy correlated with the reduced emission band-widths of BODIPY and BET dye primers and the more uniform signal intensities of BET dye primers. However, comparing % coefficients of variation (CV) for the three dye primer sets, revealed that BODIPY dye primers gave better precision than both BET and fluorescein/rhodamine dye primer sets. Several sequence-dependent motifs were identified that showed specific nucleotide-biased incorporation and were determined to be the major variable component of the total %CV. Taken together, these results show that BODIPY and BET direct DNA sequencing can accurately and precisely characterize complex mixed-base populations.

Factors influencing the emergence of resistance to indinavir: role of virologic, immunologic, and pharmacologic variables.

A major problem with the use of human immunodeficiency virus type 1 (HIV-1) protease inhibitors as monotherapy has been an unacceptably high rate of emergence of resistance. To examine possible influences on the time to emergence of resistance, 24-week data were examined from five studies in which indinavir had been administered as monotherapy or as a component of combination therapy. Monotherapy data indicated a correlation between the level of HIV-1 RNA achieved and the risk of emergence of resistance: the lower the level, the lower the risk. When combination and monotherapy regimens were compared, the group receiving indinavir + lamivudine + zidovudine had a significantly lower risk of resistance, even after adjusting for the minimum HIV-1 RNA level achieved. The findings indicate that if at all possible, HIV-1-infected patients should receive combination chemotherapy to minimize the emergence of resistance to the protease inhibitor portion of the regimen. The goal of therapy should be to decrease the HIV-1 RNA load to a less-than-detectable level.

Leptin gene therapy and daily protein administration: a comparative study in the ob/ob mouse.

We have compared the efficacy of daily injection of recombinant leptin protein (rh-leptin) with adenovirus-mediated delivery of the murine or human leptin gene (Ad-leptin) for treatment of obesity in the obese (ob/ob) mouse model. We demonstrate an improved correction profile for obesity and associated surrogate markers using the adenovirus delivery method. Rate of weight loss and percentage satiety were significantly greater in the mice treated with Adleptin. These findings were associated with lower peak serum leptin levels with Ad-leptin (22.9 +/- 2.6 ng/ml for the human gene, and 48.9 +/- 11.5 ng/ml for the murine gene) compared to rh-leptin (385.2 +/- 36.0 ng/ml). (Values are given as mean +/- standard error of the mean.) Importantly rh-leptin and ex vivo-expressed Ad-leptin were equivalently active in a functional cell-based assay. The primary difference in the two therapeutic approaches is the continuous chronic secretion of leptin mediated by gene delivery, versus the intermittent bolus delivery and rapid clearance of the daily injection of rh-leptin protein. Thus, in vivo findings suggest that leptin effects are better achieved at lower steady-state levels, a pharmacological feature attained here by gene therapy. These findings may have implications for the potential use of leptin in the treatment of obesity.

Effects of anesthesia and open-thorax surgery on coronary vascular reserve in swine.

The coronary reactive hyperemic response was examined in seven pigs under anesthetized and conscious conditions, (i.e., 5 days and 3 and 5 weeks after surgery). Tygon catheters were inserted in the descending aorta of five pigs; transonic flow probes and hydraulic occluders were placed on the left cranial descending and/or left circumflex coronary arteries. Two pigs underwent long-term implantation of similar instruments. The coronary reactive hyperemic response, expressed as repayment of flow deficit, was induced by brief complete coronary artery occlusion for 15 sec. Baseline mean arterial pressure, heart rate, and coronary blood flow were similar in the anesthetized and conscious pigs. There was also no significant difference in repayment of flow deficit between the anesthetized and conscious pigs 5 days after surgery. The repayment of flow deficit (709 +/- 144%) in conscious pigs 5 days after surgery tended to be greater, but was not statistically significant from that observed in the anesthetized pigs (510 +/- 79%). However, at 3 and 5 weeks after surgery, the reactive hyperemic flow and the repayment of flow deficit were numerically greater than those values observed in anesthetized pigs. The difference in reactive hyperemic flow between conscious and anesthetized pigs was statistically significant at week 3. The difference in repayment of flow deficit between conscious and anesthetized pigs was statistically significant at week 5. These results suggest that anesthesia, as well as recent surgery, attenuates coronary vascular reserve. The major factor in the attenuation of coronary reserve appears to be recent surgical manipulation, because repayment of flow deficit was still depressed in conscious pigs during the early phase of recovery from surgery.

Genetic correlates of in vivo viral resistance to indinavir, a human immunodeficiency virus type 1 protease inhibitor.

Indinavir (IDV) (also called CRIXIVAN, MK-639, or L-735,524) is a potent and selective inhibitor of the human immunodeficiency virus type 1 (HIV-1) protease. During early clinical trials, in which patients initiated therapy with suboptimal dosages of IDV, we monitored the emergence of viral resistance to the inhibitor by genotypic and phenotypic characterization of primary HIV-1 isolates. Development of resistance coincided with variable patterns of multiple substitutions among at least 11 protease amino acid residues. No single substitution was present in all resistant isolates, indicating that resistance evolves through multiple genetic pathways. Despite this complexity, all of 29 resistant isolates tested exhibited alteration of residues M-46 (to I or L) and/or V-82 (to A, F, or T), suggesting that screening of these residues may be useful in predicting the emergence of resistance. We also extended our previous finding that IDV-resistant viral variants exhibit various patterns of cross-resistance to a diverse panel of HIV-1 protease inhibitors. Finally, we noted an association between the number of protease amino acid substitutions and the observed level of IDV resistance. No single substitution or pair of substitutions tested gave rise to measurable viral resistance to IDV. The evolution of this resistance was found to be cumulative, indicating the need for ongoing viral replication in this process. These observations strongly suggest that therapy should be initiated with the most efficacious regimen available, both to suppress viral spread and to inhibit the replication that is required for the evolution of resistance.

Neointimal thickening after severe coronary artery injury is limited by a short-term administration of a factor Xa inhibitor. Results in a porcine model.

BACKGROUND: Fibrin- and platelet-rich thrombus formations occur as the initial event after percutaneous transluminal coronary angioplasty. We therefore tested the hypothesis that short-term administration of the recombinant tick anticoagulant peptide (rTAP), a factor Xa inhibitor, would reduce the thickness of neointima at 28 days after injury in a porcine coronary balloon angioplasty model. METHODS AND RESULTS: Continuous intravenous infusion of rTAP (average dose, 194 micrograms . kg-1 . min-1) or placebo (vehicle only) was given to the study pigs for 60 hours. The goal of anticoagulation was to maintain the activated clotting time at 200 seconds. A central venous catheter was inserted 2 days before the procedure. On the day of coronary injury, the animals were administered boluses of rTAP (6.5 mg) and then underwent injury with an oversized metallic coil by standard methods in the right, circumflex, or left anterior descending coronary artery. No significant difference in vascular injury between rTAP and vehicle control was observed after euthanasia at 28 days. Significantly less neointimal thickening occurred in the rTAP-treated animals (thickness, mean +/-SD: 0.30 +/-0.08 mm) compared with the control (0.48 +/- 0.12 mm, P< .001). CONCLUSIONS: The specific factor Xa inhibitor rTAP, when given in fully anticoagulant doses for a short duration after coronary artery injury in the porcine model, resulted in a long-term decrease in neointimal thickness. These results implicate thrombin generation in neointimal formation and suggest that administration of a potent antithrombotic for several days immediately after the procedure may influence the long-term outcome of the coronary injury with a decrease in neointimal formation.

Effects of subtype-selective and balanced angiotensin II receptor antagonists in a porcine coronary artery model of vascular restenosis.

BACKGROUND: Numerous studies have demonstrated the ability of angiotensin II (Ang II) receptor antagonists and angiotensin-converting enzyme (ACE) inhibitors to inhibit intimal hyperplasia after balloon dilation of noncoronary arteries in small-animal models, suggesting an important role for Ang II in the response to injury. Although ACE inhibitors have not been similarly effective in nonhuman coronary models or in human restenosis trials, questions remain regarding the efficacy ACE inhibitors against tissue ACE and the contributions of ACE-independent pathways of Ang II generation. Unlike ACE inhibitors, Ang II receptor antagonists have the potential to inhibit responses to Ang II independent of its biosynthetic origin. METHODS AND RESULTS: In separate studies, three Ang II receptor antagonists, including AT1 selective (L-158,809), balanced AT1/AT2 (L-163,082), and AT2 selective (L-164,282) agents, were evaluated for their ability to inhibit vascular intimal thickening in a porcine coronary artery model of vascular injury. Preliminary studies in a rat carotid artery model revealed that constant infusion of L-158,809 (0.3 or 1.0 mg X kg-1 X d-1) reduced the neointimal cross-sectional area by up to 37% measured 14 days after balloon dilatation. In the porcine studies, animals were treated with vehicle or test compound beginning 2 days before and extending 28 days after experimental angioplasty. Left anterior descending, left circumflex, and/or right coronary arteries were injured by inflation of commercially available angioplasty balloons with placement of coiled metallic stents. Infusion of L-158,809 (1 mg X kg-1 X d-1), L-163,082 (1 mg X kg-1 X d-1), or L-164,282 (1.5 mg X kg-1 X d-1) in the study animals yielded plasma drug levels sufficient either to chronically block or, for L-164,282, to spare pressor responses to exogenous Ang II. Neither L-158,809, L-163,082, nor L-164,282 had statistically significant effects (P=.12, P=.75, and P=.48, respectively, compared with vehicle-treated controls) on neointimal thickness (normalized for degree of injury) measured by morphometric analysis at day 28 after angioplasty. CONCLUSIONS: These findings indicate that chronic blockade of Ang II receptors by either site-selective or balanced AT1/AT2 antagonists is insufficient to inhibit intimal hyperplasia after experimental coronary vascular injury in the pig. The results further suggest that, unlike in the rat carotid artery, Ang II is not a major mediator of intimal thickening in the pig coronary artery.

MK-507 versus sezolamide. Comparative efficacy of two topically active carbonic anhydrase inhibitors.

Topical carbonic anhydrase inhibitors MK-507 and sezolamide hydrochloride (previously known as MK-417) were compared in a double-masked, randomized, placebo-controlled study in 82 patients with bilateral primary open-angle glaucoma or ocular hypertension. MK-507 was given every 8 or 12 hours, sezolamide every 8 hours, or placebo every 8 or 12 hours for 4 days. Both drugs lowered intraocular pressure (IOP) substantially. MK-507 was somewhat more active than sezolamide, with a peak mean IOP reduction of 26.2% for MK-507 versus 22.5% for sezolamide, although the difference between the treatments was not statistically significant. These drugs may have potential in the treatment of glaucoma.

Genetic diversity in Cucumis sativus L. assessed by variation at 18 allozyme coding loci.

The genetic diversity of the U.S. Cucumis sativus L. germplasm collection [757 plant introductions (PI) representing 45 countries] was assessed using 40 enzymes which represented 74 biochemical loci. Polymorphisms were observed at 18 loci (G2dh-1, Gpi-1, Gpi-2, Gr-1, Gr-2, Idh, Mdh-1, Mdh-2, Mdh-3, Mpi-2, Pepla-2, Peppap-2, Per-4, Pgd-1, Pgd-2, Pgm-1, Pgm-3, and Skdh). Two PIs (285606 and 215589) contained alleles [G2dh-1(1) and Per-4(2), respectively] which did not occur in any other PI. Other alleles which occurred in low frequencies (in < 1% of the PIs) included Gpi-1(3), Gpi-2(3), Gr-1(3), Gr-2(1), Idh(1), Mdh-1(2), Mdh-2(1), Peppap-2(1), and Pgd-1(1). Individual loci containing more than one allele in greater than 20% of the PIs included Mpi-2, Pepla-2, Pgd-2, and Pgm-1. Multivariate analyses aided in the reduction of data (principle components), depicted relationships among PIs (cluster), and identified the most discriminating enzyme loci (Pgm-1, Pepla-2, Gr-1, Pgd-2, Mpi-2, and Skdh) (classification and regression tree).