Biodistribution and Efficacy of Targeted Pulmonary Delivery of a Protein Kinase C-δ Inhibitory Peptide: Impact on Indirect Lung Injury.

Sepsis and sepsis-induced lung injury remain a leading cause of death in intensive care units. We identified protein kinase C-δ (PKCδ) as a critical regulator of the acute inflammatory response and demonstrated that PKCδ inhibition was lung-protective in a rodent sepsis model, suggesting that targeting PKCδ is a potential strategy for preserving pulmonary function in the setting of indirect lung injury. In this study, whole-body organ biodistribution and pulmonary cellular distribution of a transactivator of transcription (TAT)-conjugated PKCδ inhibitory peptide (PKCδ-TAT) was determined following intratracheal (IT) delivery in control and septic [cecal ligation and puncture (CLP)] rats to ascertain the impact of disease pathology on biodistribution and efficacy. There was negligible lung uptake of radiolabeled peptide upon intravenous delivery [<1% initial dose (ID)], whereas IT administration resulted in lung retention of >65% ID with minimal uptake in liver or kidney (<2% ID). IT delivery of a fluorescent-tagged (tetramethylrhodamine-PKCδ-TAT) peptide demonstrated uniform spatial distribution and cellular uptake throughout the peripheral lung. IT delivery of PKCδ-TAT at the time of CLP surgery significantly reduced PKCδ activation (tyrosine phosphorylation, nuclear translocation and cleavage) and acute lung inflammation, resulting in improved lung function and gas exchange. Importantly, peptide efficacy was similar when delivered at 4 hours post-CLP, demonstrating therapeutic relevance. Conversely, spatial lung distribution and efficacy were significantly impaired at 8 hours post-CLP, which corresponded to marked histopathological progression of lung injury. These studies establish a functional connection between peptide spatial distribution, inflammatory histopathology in the lung, and efficacy of this anti-inflammatory peptide.

Pulmonary endothelial protein kinase C-delta (PKCδ) regulates neutrophil migration in acute lung inflammation.

Excessive neutrophil migration across the pulmonary endothelium into the lung and release of oxidants and proteases are key elements in pathogenesis of acute lung injury. Previously, we identified protein kinase C-delta (PKCδ) as an important regulator of proinflammatory signaling in human neutrophils and demonstrated that intratracheal instillation of a TAT-conjugated PKCδ inhibitory peptide (PKCδ-TAT) is lung protective in a rat model of sepsis-induced indirect pulmonary injury (cecal ligation and puncture). In the present study, intratracheal instillation of this PKCδ inhibitor resulted in peptide distribution throughout the lung parenchyma and pulmonary endothelium and decreased neutrophil influx, with concomitant attenuation of sepsis-induced endothelial ICAM-1 and VCAM-1 expression in this model. To further delineate the role of PKCδ in regulating neutrophil migration, we used an in vitro transmigration model with human pulmonary microvascular endothelial cells (PMVECs). Consistent with in vivo findings, inhibition of PMVEC PKCδ decreased IL-1ß-mediated neutrophil transmigration. PKCδ regulation was stimulus-dependent; PKCδ was required for transmigration mediated by IL-1ß and fMLP (integrin-dependent), but not IL-8 (integrin-independent). PKCδ was essential for IL-1ß-mediated neutrophil adherence and NF-κB-dependent expression of ICAM-1 and VCAM-1. In PMVECs, IL-1ß-mediated production of ROS and activation of redox-sensitive NF-κB were PKCδ dependent, suggesting an upstream signaling role. Thus, PKCδ has an important role in regulating neutrophil-endothelial cell interactions and recruitment to the inflamed lung.

Use of a high caloric liquid meal as an alternative to a solid meal for gastric emptying scintigraphy.

BACKGROUND: Gastric emptying scintigraphy (GES) using a solid meal is often recommended for the evaluation of gastroparesis. However, some patients cannot tolerate the standardized egg-white sandwich (EWS) solid meal and an alternative meal is needed. AIM: The aim of this study was to compare GES, regional gastric emptying, and gastric contractility using a liquid nutrient meal (LNM; Ensure Plus(®)) to those using EWS. METHODS: Twenty healthy volunteers underwent GES using EWS and LNM on separate days. Gastric emptying was measured using scintigraphy and with a wireless motility capsule (WMC; SmartPill(®)). RESULTS: The gastric emptying half-time with LNM was similar to that with EWS (1.41 ± 0.11 vs 1.52 ± 0.08 h; P = 0.28) and the two were significantly correlated (r = 0.53; P = 0.017). There were time-related differences in gastric emptying of the LNM compared to EWS: in the first hour, gastric retention of EWS was slightly greater than that of LNM, whereas at 3 and 4 h, gastric retention of EWS was slightly less than that of LNM. Regionally, the percentage retention of the meal in the proximal stomach was greater for EWS than for LNM at 0.5 h. WMC gastric emptying times and gastric contractility for the two meals were not significantly different. CONCLUSIONS: Overall gastric emptying of the LNM was similar to that of the EWS meal. The LNM empties without a lag phase and takes slightly longer to empty from the distal stomach, likely due to its higher fat content. These differences are likely due to early accommodation with retention of solids in the proximal stomach and the need for trituration of solids. We conclude that this LNM can serve as an alternative to the conventional solid EWS for GES.

Update on gastrointestinal radiopharmaceuticals and dosimetry estimates.

The gold standard technique for measuring gastric emptying is scintigraphy using radiolabeled test meals. Recently, a standardized radiolabeled solid meal has been proposed and adopted by many centers. There is still a need for alternative meals, and several such meals with demonstrated radiolabel stability have been evaluated in small numbers of subjects. Updated radiation dosimetry associated with these meals has been calculated for adult males and adult females with normal gastrointestinal transit as well as transit abnormalities.

Binding and Internalization of Iron Oxide Nanoparticles Targeted to Nuclear Oncoprotein.

A targeted nanoconjugate is being developed for non-invasive detection of gene expression in cells expressing the JC virus oncoprotein, T-antigen, which has been associated with medulloblastoma and other cancers. JC virus T-antigen localizes predominantly to the nucleus via a classical monopartite nuclear localization signal (NLS). An antibody fragment which recognizes JC virus T-antigen was attached to cross-linked dextran coated iron oxide nanoparticles. Radiolabeled conjugates were added to mouse medulloblastoma cells expressing the target T-antigen to test their ability to bind to tumor cells and be internalized by the cells. All conjugates containing targeting antibody bound to cells and were internalized, with increasing levels over time. There was no difference in cell binding or internalization among conjugates containing 2, 4, 6 or 8 antibody fragments per nanoparticle. Conjugates with only nonspecific antibody on nanoparticles, or unconjugated nonspecific antibody, had significantly lower total binding and internalization than conjugates with targeting antibody. Unconjugated targeting antibody had equivalent or lower cell uptake compared with targeted nanoparticle conjugates. Specificity of uptake was demonstrated by >80% reduction of nanoconjugate uptake in the presence of 100 fold excess of unconjugated antibody. The presence of a membrane translocation peptide (Tat) on the nanoparticles in addition to targeting antibody did not improve nanoconjugate internalization over the internalization caused by the antibody alone. This antibody nanoconjugate demonstrates feasibility of targeting a nuclear protein and suggests that a minimum number of antibody fragments per nanoparticle are sufficient for achieving binding specificity and efficient uptake into living cells.

Radiation-guided targeting of combretastatin encapsulated immunoliposomes to mammary tumors.

PURPOSE: Radiation upregulates expression of endothelial cell adhesion molecules providing a potential avenue for targeting drugs to irradiated tissue. Induced upregulation of E-selectin can be used to target immunoliposomes to solid tumors. The effects of targeting immunoliposomes containing the antivascular drug combretastatin disodium phosphate (CA4P) to irradiated mammary tumors were investigated in this study. METHODS: Mice bearing transplanted MCa-4 mouse mammary tumors were assigned to one of the factorial treatments permuting the administration of free CA4P, tumor irradiation, CA4P encapsulated liposomes, and CA4P encapsulated immunoliposomes (conjugated with anti-E-selectin). Single and fractionated dosing of radiation and/or CA4P was evaluated. RESULTS: For single dose treatments the group that received a single dose of radiation plus a single dose of immunoliposomes showed a significant delay in tumor growth compared to all other treatment groups. Fractionated radiation plus fractionated doses of immunoliposomes resulted in further tumor growth delay; however, it was not significantly different from other fractionated dose treatment groups that combined radiation and CA4P. CONCLUSIONS: Targeting of antivascular drugs to irradiated tumors via ligand-bearing liposomes results in significant tumor growth delay. This effect can be further potentiated using a fractionated irradiation dosing schedule combined with fractionated immunoliposome treatments.

Transsphincteric pH profile at the gastroesophageal junction.

BACKGROUND & AIMS: Esophageal acid exposure conventionally is measured 5 cm above the lower esophageal sphincter (LES). The aim of this study was to compare pH profiles at sites within the LES, the distal esophagus, and the proximal stomach. METHODS: Ten normal subjects underwent esophageal manometry followed by 24-hour esophagogastric pH monitoring using an 8-channel pH probe recording at 5 and 1.5 cm above and at 0, 1.5, 3.0, 4.5, 6.0, and 9.5 cm below the proximal LES border. During pH recording, a 4-hour gastric emptying test with an egg sandwich meal was performed. RESULTS: The LES was 3.2 +/- 0.4 cm in length. There was a progressive increase in acid exposure from the esophageal to the gastric pH sensors. pH was less than 4 for 3.4% +/- 1.6%, 12.7% +/- 8.5%, 26.5% +/- 10.2%, 48.1% +/- 11.3%, 66.5% +/- 9.9%, 80.8% +/- 5.6%, 89.2% +/- 3.0%, and 96.7% +/- 1.1% of the total time for pH probes at 5 and 1.5 cm above and 0, 1.5, 3, 4.5, 6.0, and 9.5 cm below the proximal LES border, respectively. Percentage acid exposures correlated significantly with the position of the probe (r = -0.95; P < .01). Intrasphincteric acidity increased postprandially. Gastric emptying was correlated inversely with the intragastric hydrogen ion concentration (r = -0.82). CONCLUSIONS: The percentage of recording time that pH was less than 4 was significantly higher in the intrasphincteric area and 1.5 cm above the proximal LES compared with the traditional site 5 cm above the proximal manometric LES border. High acid exposure in the intrasphincteric region might explain the susceptibility of the distal esophagus to erosions, strictures, and Barrett's esophagus.

Egg labeling methods for gastric emptying scintigraphy are not equivalent in producing a stable solid meal.

UNLABELLED: A wide range of radiolabeled test meals have been used for gastric emptying scintigraphy. The purpose of this study was to test whether (99m)Tc-sulfur colloid-labeled liquid egg white is as stable as 2 fresh whole eggs labeled with (99m)Tc-sulfur colloid and whether the cooking method is important. METHODS: Whole eggs and liquid egg white were mixed with (99m)Tc-sulfur colloid and cooked by either microwaving or frying on a griddle. The cooked eggs were tested for breakdown after 2 and 4 h of incubation in gastric fluid or HCl. RESULTS: Labeled liquid egg white, prepared by either method of cooking, exhibited less breakdown in gastric fluid than whole eggs. Whole eggs cooked in the microwave exhibited significantly more breakdown than liquid egg white. CONCLUSION: (99m)Tc-Sulfur colloid binds better to egg whites compared with whole eggs. These results emphasize the need to evaluate the stability of new radiolabeled test meal preparations, including the method of cooking.

Platelet binding and biodistribution of [99mTc]rBitistatin in animal species and humans.

INTRODUCTION: 99mTc recombinant bitistatin (rBitistatin) is a radioligand for alphaIIbbeta3 (glycoproteins IIb/IIIa) receptor on platelets and is being developed as a diagnostic radiopharmaceutical for in vivo imaging of acute thrombi and emboli. Prior to the first administration of [99mTc]rBitistatin to human subjects, its biodistribution and effects on platelets were evaluated in animals. This paper reports findings in animal studies in comparison with initial findings in normal human subjects. METHODS: [99mTc]rBitistatin was administered to mice, guinea pigs and dogs to assess time-dependent organ distribution, urinary excretion and blood disappearance rates. Blood samples were analyzed to determine radioligand binding to circulating platelets and the extent of plasma protein binding. The effect of [99mTc]rBitistatin on circulating platelet count was determined. These factors were also determined in normal human subjects who received [99mTc]rBitistatin as part of a Phase I clinical trial. RESULTS: The main organs that accumulated [99mTc]rBitistatin were kidneys, liver and spleen in all animal species and humans. The main organs seen on human images were the kidneys and spleen. Liver uptake was fainter, and soft-tissue background was low. [99mTc]rBitistatin bound to circulating platelets in blood, with a higher percentage of binding to platelets in guinea pigs and dogs compared to that in humans. Plasma protein binding was low and of little consequence in view of platelet binding. The main route of excretion was through the urine. [99mTc]rBitistatin did not affect platelet counts in humans or dogs. CONCLUSIONS: [99mTc]rBitistatin, when administered at low doses for imaging, has no adverse effects on platelets and has the qualitative biodistribution predicted by animal studies. [99mTc]rBitistatin was found to bind to circulating platelets in humans, suggesting that it will be able to bind to activated platelets in vivo in patients with acute thrombi.

Differences in binding of (99m)Tc-disintegrins to integrin alphavbeta3 on tumor and vascular cells.

UNLABELLED: Disintegrins, which contain an Arg-Gly-Asp sequence in their binding domains are antagonists of integrins such as alphavbeta3. The purpose of this study was to compare a range of disintegrins with different integrin selectivities for their binding behavior in vitro to vascular endothelial cells bearing alphavbeta3 and to cultured tumor cells which express alphavbeta3. METHODS: Five disintegrins (bitistatin, kistrin, flavoridin, VLO4 and echistatin) and a cyclic pentapeptide, c[RGDyK], were radiolabeled with (99m)Tc and tested for binding to cells in vitro. RESULTS: (99m)Tc-Kistrin, flavoridin and VLO4 had the highest binding, (99m)Tc-echistatin had moderate binding, and (99m)Tc-bitistatin and (99m)Tc-c[RGDyK] had low binding to cells. The observed binding was attributed to alphavbeta3 to various extents: echistatin, bitistatin>kistrin>flavoridin>VLO4. Cancer cells internalized bound disintegrins after binding, but endothelial cells did not. After binding to endothelial cells, (99m)Tc-kistrin was not displaced by competing peptide or plasma proteins. CONCLUSIONS: These data suggest that radiolabeled kistrin, flavoridin and VLO4 may have advantages over labeled bitistatin and small cyclic peptides for targeting alphavbeta3 in vivo. Since receptor-bound radioligand is not internalized by endothelial cells, disintegrins may provide an advantage for targeting alphavbeta3 on vasculature because they bind strongly to surface receptors and are not readily displaced.

Functional expression of bitistatin, a disintegrin with potential use in molecular imaging of thromboembolic disease.

Bitistatin is a single-chain disintegrin which contains 83 amino acids and is internally crosslinked with seven disulfide bonds. This platelet aggregation inhibitor, which binds with high affinity to the alphaIIbbeta3 integrin, has potential use as the basis for a radiotracer to locate thrombi and emboli by scintigraphic imaging. A method amenable to large-scale, consistent production of bitistatin was sought. A synthetic gene coding for bitistatin was inserted into two different Escherichia coli expression vectors. One vector expressed recombinant bitistatin (rBitistatin) as a cleavable fusion protein and the other expressed rBitistatin as an isolated protein. In both cases, rBitistatin contained an additional amino acid (Gly) at the N-terminus compared with the native protein. The fusion protein was purified by affinity chromatography, then cleaved enzymatically to release rBitistatin, which was purified by reversed-phase high performance liquid chromatography (HPLC) to a single active form. The rBitistatin produced as an isolated protein was purified from cell lysate by HPLC in a reduced form, then refolded, and purified again by HPLC. Yields of active rBitistatin averaged 12 mg/L for expression as an isolated protein, 10 times as high as when the fusion protein was employed. Structural assays confirmed the expected mass and sequence of the product. Functional assays (inhibition of platelet aggregation in vitro, equilibrium binding to platelets in vitro, and binding of labeled protein to experimental thrombi and emboli in vivo) confirmed that rBitistatin retained the functional characteristics of native bitistatin.

Evaluation of 64Cu- and 125I-radiolabeled bitistatin as potential agents for targeting alpha v beta 3 integrins in tumor angiogenesis.

The formation of new blood vessels (angiogenesis) is a feature common to all solid tumors. The integrin receptor alpha(V)beta(3), which is found on endothelial cells lining newly growing blood vessels at a higher density than on mature blood vessels, is being explored as a marker for tumor angiogenesis. Bitistatin, a member of the disintegrin family of polypeptides, has affinity for alpha(V)beta(3) integrins. To determine whether radiolabeled bitistatin could target tumors, its biodistribution was tested in tumor-bearing mice. For initial validation studies, (125)I-bitistatin was injected into BALB/c mice bearing EMT-6 mouse mammary carcinoma tumors, a model that is highly vascular but which lacks alpha(V)beta(3) directly on tumor cells. Tumor uptake reached maximal values (11.7 +/- 4.6 %ID/g) at 2 h. Co-injection of 200 microg of unlabeled bitistatin reduced tumor uptake 62%, suggesting that the binding of (125)I-bitistatin is receptor-mediated. This work was extended to include the beta(+)-emitting radionuclide (64)Cu, which was attached to bitistatin via 1,4,7,10-tetraazacyclododecane-N,N',N' ',N' "-tetraacetic acid (DOTA). This modification did not significantly alter receptor binding in vitro. MicroPET images obtained with (64)Cu-DOTA-bitistatin showed that the tumor could easily be identified 4 h after administering the radiopharmaceutical. The biodistribution of (64)Cu-DOTA-bitistatin differed from the (125)I analogue, in that maximum tumor uptake was nearly 8-fold lower and took at least 6 h to reach maximal binding (1.6 +/- 0.2 %ID/g). As with (125)I-labeled bitistatin, the (64)Cu conjugate showed a 50% reduction in tumor uptake with the co-injection of 200 microg of unlabeled bitistatin (0.8 +/- 0.2 %ID/g). Competition studies with integrin-specific peptides indicated that the tumor uptake was related to both alpha(v)beta(3) and alpha(IIb)beta(3) integrin binding. To see if tumor uptake could be improved upon, (64)Cu was tethered to bitistatin using bromoacetamidobenzyl-1,4,7,10-tetraazacyclododecane-N,N',N' ',N' "-tetraacetic acid (BAD). Tumor uptake for (64)Cu-BAD-2IT-bitistatin was higher than the DOTA conjugate at all time points, reaching a maximum at least 6 h postinjection (5.2 +/- 0.6 %ID/g); however, this was accompanied by higher uptake in nontarget organs at all time points. Radiolabeled ligands of this type may be useful in the targeting of tumor angiogenesis, but the choice of radiolabeling approach has a significant impact on the in vivo properties of the radioligand.

Design and synthesis of a short-chain bitistatin analogue for imaging thrombi and emboli.

Previously, we showed that labeled bitistatin analogues possessed excellent characteristics for imaging both deep-vein thrombosis and pulmonary embolism. We hypothesized that the N-terminal amino acid sequence of bitistatin, which is different from other disintegrins, likely interacts with the binding site of platelets to confer desirable properties to bitistatin for imaging. In this study, we present the design, synthesis, and initial biological testing of a short-chain analogue of the native 83-amino-acid bitistatin sequence. Our initial molecular modeling of the binding loop of bitistatin showed that the minimal sequence that represented the binding region was a cyclic 10 amino acid sequence cyclo[Cys-Arg-Ile-Ala-Arg-Gly-Asp-Trp-Asn-Cys(S)]. Systematic modeling of a truncated N-terminal sequence of bitistatin fused with the optimized binding region having a thioether sequence through a Gaba spacer ultimately yielded the 24-amino acid peptide, cyclo-[CH(2)CO-Arg-Ile-Ala-Arg-Gly-Asp-Trp-Asn-Cys(S-)]-Gaba-Gly-Asn-Glu-Ile-Leu-Glu-Gln-Gly-Glu-Asp-Ser-Asp-Ser-Lys-OH, 1. The peptide was then coupled to the hydrazino-nicotinic acid bifunctional chelating agent and the purified adduct labeled with (99m)Tc using tricine as a coligand. Binding of the unlabeled and labeled peptide to stimulated human platelets was assayed in vitro. The (99m)Tc labeling yield was > 90%. The in vitro binding assays showed that the IC(50) for inhibition of platelet aggregation was 3694 nM, while the Kd of the (99m)Tc labeled peptide was 185 nM, indicating moderate affinity for the receptor. The (99m)Tc-labeled peptide was able to identify sites of experimental thrombi and emboli in a canine model. The results suggest initial success in attempting to mimic the behavior of bitistatin for imaging thrombi and emboli.

Simultaneous assessment of gastric accommodation and emptying: studies with liquid and solid meals.

UNLABELLED: The aim of this study was to develop a scintigraphic test to measure gastric emptying and accommodation simultaneously. METHODS: Gastric emptying and accommodation were measured in healthy subjects. To determine gastric accommodation, the stomach was imaged with SPECT 20 min after intravenous administration of 185 MBq (5 mCi) (99m)Tc-pertechnetate. After ingestion of 11 MBq (300 micro Ci) (111)In-diethylenetriaminepentaacertic acid in a liquid nutrient drink or an (111)In-oxine-labeled egg sandwich, dual-isotope imaging assessed SPECT gastric dimensions and gastric emptying every 20 min up to 240 min. Gastric accommodation was calculated as the percentage change in planar (2-dimensional) gastric cross-sectional area (CSA) using a left anterior oblique planar projection and the percentage change in total SPECT gastric voxel counts (3-dimensional) compared with the baseline image. RESULTS: With the liquid nutrient drink (9 subjects), maximal mean CSA (158% +/- 12% of baseline; P < 0.05) occurred 40 min after meal ingestion, when only 69% +/- 3% of the radiolabeled liquid nutrient drink remained in the stomach. At 120 min, mean CSA was 125% +/- 8% of baseline, but only 35% +/- 3% of the liquid nutrient drink remained in the stomach. Using SPECT to measure 3-dimensional volumes, maximal gastric volume occurred 20 min after meal ingestion (189% +/- 25% of baseline). With the solid egg meal (10 subjects), maximal total CSA (159% +/- 13% of baseline) occurred immediately after meal ingestion; total CSA remained significantly increased above baseline for the first 3 h after ingestion of the egg meal, despite only 12% +/- 4% gastric retention at 3 h. Using SPECT to measure 3-dimensional volumes, maximal gastric volume occurred immediately after the meal (184% +/- 19% of baseline). CONCLUSION: This method permits simultaneous measurement of gastric emptying and accommodation. In healthy subjects, the gastric accommodation response is prolonged and persists despite nearly complete emptying of a liquid or solid meal.

Effect of altering gastric emptying on postprandial plasma glucose concentrations following a physiologic meal in type-II diabetic patients.

The purpose of this study was to determine the effects of altering gastric emptying on postprandial plasma glucose concentration after a physiologic meal in patients with type II diabetes mellitus (T II DM). Nine T II DM patients underwent a double-blind, randomized, three-way crossover study, receiving erythromycin 200 mg, morphine 8 mg, or normal saline (placebo) intravenously prior to ingestion of a radiolabeled, dual-isotope, solid-liquid meal. Gastric emptying of solids and liquids and serial plasma glucose, glucagon, and serum insulin concentrations were measured at baseline and for 5 hr after meal ingestion. Erythromycin accelerated and morphine delayed solid- and liquid-phase gastric emptying compared to placebo (P < 0.05). During the first hour, the postprandial plasma glucose concentrations were higher after erythromycin (P < 0.05) and lower after morphine (P < 0.05) compared to placebo. The peak postprandial plasma glucose concentration was higher after erythromycin (P = 0.05) and lower after morphine (P < 0.05) compared to placebo. In conclusion, pharmacologic acceleration of gastric emptying resulted in higher postprandial glucose concentrations, while delaying gastric emptying resulted in lower postprandial glucose concentrations after a physiologic meal in T II DM. These results suggest that administration of opiate analgesics or prokinetic agents to diabetic patients may alter glucose control. Modifying gastric emptying may be helpful in achieving glucose control in T II DM.

Treatment of idiopathic gastroparesis with injection of botulinum toxin into the pyloric sphincter muscle.

OBJECTIVES: We aimed to determine if botulinum toxin injection into the pyloric sphincter improves gastric emptying and reduces symptoms in patients with idiopathic gastroparesis. METHODS: Patients with idiopathic gastroparesis not responding to prokinetic therapy underwent botulinum toxin (80-100 U, 20 U/ml) injection into the pyloric sphincter. Gastric emptying scintigraphy was performed before and 4 wk after treatment. Total symptom scores were obtained from the sum of eight upper GI symptoms graded on a scale from 0 (none) to 4 (extreme). RESULTS: Ten patients were entered into the study. The mean percentage of solid gastric retention at 4 h improved from 27+/-6% (normal < 10%) before botulinum toxin injection into the pylorus to 14+/-4% (p = 0.038) 4 wk after treatment. The symptom score decreased from 15.3+/-1.7 at baseline to 9.0+/-1.9 (p = 0.006) at 4 wk, a 38+/-9% decrease. Improvement in symptoms tended to correlate with improved gastric emptying of solids (r = 0.565, p 0.086). CONCLUSIONS: This initial pilot study suggests that botulinum toxin injection into the pylorus in patients with idiopathic gastroparesis improves both gastric emptying and symptoms.

Simultaneous measurement of gastric emptying with a simple muffin meal using [13C]octanoate breath test and scintigraphy in normal subjects and patients with dyspeptic symptoms.

The objective of this study was to determine how the [13C]octanoate breath test (OBT) using a muffin meal correlates with gastric emptying scintigraphy (GES) in normal subjects and patients with dyspeptic symptoms. Ten normal subjects and 23 patients with dyspeptic symptoms underwent simultaneous GES and [13C]OBT. After an overnight fast, a muffin labeled with [99mTc]-sulfur colloid and [13C]octanoate was ingested along with water labeled with [111In]DTPA. Breath samples and scintigraphic images were obtained at baseline and at regular postprandial intervals over 6 hr. In the normal subjects, the mean GES 71/2 of solids and liquids were 64 +/- 17 and 55 +/- 27 minutes, respectively. The calculated OBT T1/2 using the 6-hr breath collection was 138 +/- 15 min and correlated with T1/2 for solids by GES (r = 0.664; P = 0.051), but did not correlate with T1/2 for liquids by GES (r = 0.13; P = 0.738). In dyspeptic patients, the T1/2 for GES was 87 +/- 53 min and 81 +/- 70 min for solids and liquids, respectively. The mean OBT T1/2 was 155 +/- 57 min and correlated with GES T1/2 for solids (r = 0.86; P < 0.001) and GES T1/2 for liquids (r = 0.73; P < 0.001). Delayed gastric emptying (GE) of the muffin meal was identified by scintigraphy in seven patients. The sensitivity and specificity for OBT identifying delayed GE were 86% and 94%. Use of the initial truncated 4-hr OBT results also revealed a significant correlation between OBT and GES T1/2 for solids (r = 0.86; P < 0.001) with sensitivity and specificity for detecting delayed GE of 86% and 94%, respectively. In addition, a linear regression model was able to reduce the number of collection points to four, while maintaining the same sensitivity and specificity. In conclusion, the OBT for GE, using an easily prepared muffin meal, significantly correlates with GES for solids. This muffin-based OBT is a sensitive and specific method to detect delayed GE in dyspeptic patients.