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Biomedical subjects

S Cleary

Publications and source records attributed to S Cleary.

17 recordsLinked to original sources
PubMed1991

Thermodynamics of ligand binding and denaturation for His64 mutants of tissue plasminogen activator kringle-2 domain.

The contribution of His64 to the function and stability of tissue plasminogen activator (t-PA) kringle-2 domain (His244 in t-PA numbering) has been studied by using microcalorimetric methods to compare the ligand binding and thermal denaturation behavior of wild-type kringle-2 and mutants having His64 replaced with Tyr or Phe. This site was examined because modeling studies suggested that the His64 side chain could play an important role in ligand binding by forming an ion-pair with the carboxylate of the ligand, L-lysine. Kringle-2 domains were expressed by secretion of the 174-263 portion of t-PA in E. coli and purified as previously described for the wild-type domain. Both mutant proteins retain affinity for L-lysine, although reduced three- to four-fold relative to wild-type, demonstrating that His64 does not interact with the ligand carboxylate through an ion-pair interaction or by hydrogen bonding. The H64Y substitution does result in an altered specificity of the lysine binding site with the mutant domain having greatest affinity for a ligand of 6.8 A chain length, whereas the wild-type domain prefers an 8.8 A long ligand. For both wild-type and mutant, the binding of the optimal chain length ligand is dominated by enthalpic effects (delta H = -6,000 to -7,000 cal/mol) and T delta S accounts for less than 15% of delta G. In addition, the H64Y mutant differs from wild-type in the effect of ligand alpha-amino group modification on binding affinity. Based on examination of the x-ray structure recently determined for wild-type kringle-2, the specificity changes accompanying the H64Y substitution probably result from changes in side chain interactions in the lysine binding site. Thermal denaturation experiments show that the H64Y mutant is also more stable than the wild-type protein with the difference in stabilization free energy (delta delta G) equal to 2.7 kcal/mol at 25 degrees C and pH 3. The increased stability of the mutant appears to be related to the difference in hydrophobicity between His and Tyr.

Binding Sites↗
PubMed1989

Effect of residue 65 substitutions on thermal stability of tissue plasminogen activator kringle-2 domain.

We have used differential scanning calorimetry to measure the effect of replacements of valine 65 on thermal stability of the isolated kringle-2 domain of tissue plasminogen activator (t-PA). The role of this site in stability was examined because a human t-PA variant having this valine (residue 245 in t-PA numbering) replaced with a methionine has been described [Johnston, M.D., & Berger, H. (1987) U.K. Patent Application GB 2176702A]. Mutants of kringle-2 having valine 65 replaced with Met, Leu, Ile, Thr, Ala, or Ser were constructed by using site-directed mutagenesis in conjunction with a restricted site selection strategy. Isolated kringle-2 domains were expressed in Escherichia coli and purified as previously described for the wild-type domain [Cleary, S., Mulkerrin, M.G., & Kelley, R.F. (1989) Biochemistry 28, 1884-1891]. None of these substitutions results in a significant perturbation of the native conformation of kringle-2 as judged by far-UV circular dichroism and equilibrium dialysis measurements of L-lysine affinity. A two-state analysis of the heat capacity profile observed for heating a solution of wild-type (w-t) kringle-2 containing 100 mM citrate, pH 4.5, provides values of 64.3 +/- 0.8 degrees C for Tg (melting temperature), 81 +/- 5 kcal/mol for delta H g, and 1.2 +/- 0.9 kcal/(mol-deg) for delta C p. Thermal denaturation of w-t kringle-2 is reversible in the pH range 3-6 as indicated by the observation of similar heat capacity profiles for consecutive heating cycles and also recovery of spectroscopic and lysine binding properties upon cooling the heat-denatured protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1989

Purification and characterization of tissue plasminogen activator kringle-2 domain expressed in Escherichia coli.

We have expressed the 174-263 fragment (kringle-2 domain) of human tissue-type plasminogen activator (t-PA) in Escherichia coli by secretion into the periplasmic space using the alkaline phosphatase promoter and stII enterotoxin signal sequence. A large portion of the secreted protein is associated with an insoluble cellular fraction. This material can be solubilized by extraction with denaturant and reducing agent and then recovered in active form by refolding in the presence of reduced and oxidized glutathione. Kringle-2 is then easily purified by affinity chromatography on lysine-Sepharose followed by cation-exchange chromatography. The isolated protein has an amino acid composition and N-terminal sequence as expected for the 174-263 fragment of t-PA, indicating that the signal peptide has been properly removed. Circular dichroic spectra suggest that the protein is folded similar to the kringle-4 domain of plasminogen [Castellino et al. (1986) Arch. Biochem. Biophys. 247, 312-320]. Equilibrium dialysis experiments indicate a single binding site on kringle-2 for L-lysine having a KD of 100 microM. Using a method based on elution of kringle from lysine-Separose with omega-aminocarboxylic acids [Winn et al. (1980) Eur. J. Biochem. 104, 579-586], we have shown the lysine binding site of t-PA kringle-2 to have a preference for a ligand with 8.8-A separation between amine and carboxylate functions. Charge interactions with the epsilon-amino group of L-lysine are important in binding since the affinities for N epsilon-acetyl-L-lysine, L-arginine, and gamma-guanidinobutyric acid are decreased greater than 2000-fold, 200-fold, and 12-fold, respectively, relative to the affinity for L-lysine.(ABSTRACT TRUNCATED AT 250 WORDS)

Cloning, Molecular↗
PubMed1988

Pharmacokinetics of intraperitoneally administered dipyridamole in cancer patients.

The pharmacokinetics of i.p. administered dipyridamole was studied in six patients to explore the feasibility of using this drug as a modulator of antimetabolite activity in extravascular spaces. Infusions of dipyridamole (50 mg/m2 in 2 liters of normal saline) into the peritoneal cavity resulted in peak drug concentrations 5 to 20 times higher in that cavity than in the plasma. The peritoneal decay data for dipyridamole fitted very well to a single compartment open pharmacokinetic model with one exponential term, while the plasma data are adequately described by a single compartment model with two exponentials (a short absorption phase). The mean peritoneal half-life for total extractable dipyridamole was 3.3 +/- 1.9 (SD) h, and the mean peritoneal clearance was 0.4 +/- 0.3 liters/h/m2. The mean plasma half-life of total dipyridamole in our patients was 2.2 +/- 1.2 h, and the mean clearance value was 5.7 +/- 4.7 liters/h/m2. The area under the concentration versus time curve was calculated to be 626 +/- 312 microM-h for the peritoneal cavity and 45 +/- 20 microM-h for the plasma. Using membrane ultrafiltration, we have measured the concentration of free (non-protein bound) dipyridamole in each patient. While the peritoneal clearance values of free and total drug are comparable, the plasma clearance of free dipyridamole was 47 +/- 39 liters/h/m2. This increased plasma clearance resulted in a plasma area under the concentration versus time curve of 8.3 +/- 5.1 microM-h, which suggests minimal systemic exposure. Our data show that instillation of dipyridamole into the peritoneal cavity resulted in much higher local drug exposure than systemic exposure, confirming the feasibility of using this drug to augment antimetabolite activity within the peritoneal cavity. Since dipyridamole is highly protein bound in the plasma but less so in the peritoneal cavity, these data imply that peritoneal exposure to active (free) dipyridamole is far greater than systemic exposure in our patients.

Aged↗
PubMed1987

Streamlined managers.

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Administrative Personnel↗
PubMed1987

Phase I trial of combination therapy of cancer with N-phosphonacetyl-L-aspartic acid and dipyridamole.

While N-phosphonacetyl-L-aspartic acid (PALA), an inhibitor of de novo pyrimidine biosynthesis, demonstrated a unique spectrum of activity during preclinical drug evaluation, multiple clinical trials have shown it to possess minimal clinical activity. One explanation for the disappointing results is the possibility that tumor cells are able to utilize circulating uridine in the synthesis of pyrimidines (salvage pathway). Dipyridamole, an inhibitor of nucleoside transport, has been demonstrated experimentally to potentiate the cytotoxicity of PALA significantly. In addition, this agent has a long safety record when used clinically in man. A phase I trial of this two-drug combination was therefore conducted, with a fixed oral dose of dipyridamole (50 mg/m2 every 6 h) and an escalating i.v. dose of PALA administered every 3 weeks. The dose-limiting toxicity with this schedule was diarrhea and abdominal cramping pain at a PALA dose of 3900-4200 mg/m2. Among the 65 patients participating in this trial 4 objective responses (2 partial, 2 minimal) were observed. Because of the potential for unique clinical synergy between PALA and dipyridamole further investigation should be considered.

Adult↗
PubMed1987

Long-term survival of advanced refractory ovarian carcinoma patients with small-volume disease treated with intraperitoneal chemotherapy.

Ninety ovarian carcinoma patients failing primary intravenous (IV) combination chemotherapy were treated with cisplatin-based combination intraperitoneal therapy. Sixty-five patients had residual disease greater than 2 cm at the start of intraperitoneal therapy. Their median survival was 8 months. Twenty-five patients had disease less than 2 cm; their median survival was greater than 49 months, and the survival curve has an apparent plateau at 69%, with no relapses having occurred after 32 months. The median survival for all 90 patients was 15 months. The median duration of follow-up for all patients was 37 months. These results confirm the critical role of tumor bulk in determining the effectiveness of intraperitoneal therapy, and suggest a role for intraperitoneal salvage treatment in patients with small-volume disease.

Actuarial Analysis↗
PubMed1986

Cisplatin administered by the intracavitary route as treatment for malignant mesothelioma.

Twenty-one patients with malignant mesothelioma were treated with an experimental intracavitary chemotherapy regimen of weekly intraperitoneal or intrapleural cisplatin (90-100 mg/m2) with simultaneous intravenous sodium thiosulfate delivered to protect against cisplatin-induced nephrotoxicity. One of eight patients (12.5%) receiving intrapleural therapy and nine of 13 patients receiving intraperitoneal therapy demonstrated objective evidence of a clinical response, including three surgically defined major tumor regressions (23%). Patients receiving intrapleural treatment had more advanced disease prior to therapy than those receiving intraperitoneal therapy. It was concluded that intraperitoneal cisplatin is an active treatment program for intra-abdominally localized mesothelioma. Additional investigation of intrapleural cisplatin should be undertaken in a patient population with less advanced disease or following surgical debulking.

Actuarial Analysis↗
PubMed1986

Hypomagnesemia following high-dose intracavitary cisplatin with systemically administered sodium thiosulfate.

Seventy-one patients receiving a minimum of two courses of high-dose intracavitary cisplatin (100-200 mg/m2/course) with i.v. thiosulfate administered to protect against cisplatin-induced renal insufficiency were retrospectively evaluated to examine the influence of thiosulfate and large cumulative doses of cisplatin on the incidence of hypomagnesemia. Only 8% of 50 patients who had normal serum magnesium levels prior to the initiation of the experimental program became hypomagnesemic during the therapeutic trial. Similarly, while 67% of the 21 patients with low initial serum magnesium levels remained hypomagnesemic, 33% had normal serum levels at the completion of therapy. It is suggested that the intravenous administration of thiosulfate might have been responsible for the strikingly low incidence of hypomagnesemia in this patient population. A prospective evaluation of the utility of sodium thiosulfate in preventing cisplatin-induced renal magnesium wasting appears indicated.

Adult↗
PubMed1986

Ip chemotherapy employing a regimen of cisplatin, cytarabine, and bleomycin.

Thirty-seven patients with refractory ovarian carcinoma or other malignancies principally confined to the peritoneal cavity were treated with a combination ip chemotherapy regimen consisting of cisplatin (100 or 200 mg/m2), cytarabine (1,200 mg/m2), and bleomycin (15 or 2 U/m2) repeated at 28-day intervals. Sodium thiosulfate was simultaneously administered iv to protect against cisplatin-induced nephrotoxicity. While only one of 18 patients (6%) with bulky residual ovarian carcinoma experienced a partial remission, two of six evaluable patients with minimal residual disease experienced surgically defined complete remissions. Local abdominal pain was often severe with the higher dose of bleomycin and was occasionally a problem with the lower dose schedule. Fever was common (greater than 70% of courses), but was reduced in degree by pretreatment with steroids. We conclude that combination ip chemotherapy with this three-drug regimen can result in objective tumor regressions and surgically defined complete remissions in patients with minimal residual ovarian carcinoma who have failed to achieve a complete remission following cisplatin-based iv chemotherapy.

Abdominal Neoplasms↗
PubMed1986

Plasma uridine changes in cancer patients treated with the combination of dipyridamole and N-phosphonacetyl-L-aspartate.

Dipyridamole (DP) and N-phosphonacetyl-L-aspartate (PALA) act synergistically in vitro against many cell lines and in vivo against human ovarian carcinoma xenografts. We have conducted a phase I clinical trial of DP p.o. (50 mg/m2, every 6 h) in combination with PALA (starting at 500 mg/m2 i.v. with 300-mg dose escalations). Sixty-five patients were entered into this study, and we have established the maximum tolerated dose of PALA to be 4.5 g/m2 when combined with DP, which is approximately 80% of the previously reported maximum tolerated dose for PALA alone. The observed toxicities of DP plus PALA were mild and were similar to those reported for PALA alone. Bone marrow toxicities were not evident at any PALA dose. Ten patients with a mean pretreatment plasma uridine concentration of 3.49 +/- 1.28 (SD) microM had their plasma uridine reduced to 2.29 +/- 0.70 microM 9 h after DP p.o. A peak plasma DP concentration of 1.86 +/- 0.99 microM was achieved approximately 2 h after p.o. dosing. Nine patients who had a reduced plasma uridine concentration of 2.46 +/- 0.61 microM after 1 week of DP had their plasma uridine further reduced by PALA to 0.87 +/- 0.23 microM 7 h post-PALA. Daily plasma uridine measurements in two patients during their DP treatment confirmed the previously described pattern for Day 1, but the data suggest a slight recovery (15%) in plasma uridine by Day 2. Daily sampling in two other patients after a single PALA dose of 4.2 g/m2 showed that their plasma uridine declined 5 h after the PALA dose and remained depressed for 6 days in one patient and 11 days in the other. These results suggest that DP worked in synergy with PALA to lower circulating uridine in cancer patients. The mechanism for the ability of DP to reduce plasma uridine is not known, but there is evidence that DP can inhibit cellular efflux of uridine as well as its uptake. DP may reduce plasma nucleoside pools in addition to blocking nucleoside salvage and therefore have general applicability in other chemotherapy regimens.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1986

Complications of extensive adhesion formation after intraperitoneal chemotherapy.

The results of modeling studies have suggested and clinical investigations have confirmed a major pharmacokinetic advantage for exposure of the peritoneal cavity to certain chemotherapeutic agents compared with exposure of the systemic circulation when these drugs are delivered through the intraperitoneal route. Objective antitumor responses and significant palliation of symptoms have been demonstrated. One major concern with the use of intraperitoneal therapy is the risk of inducing peritoneal sclerosis and extensive adhesion formation. At the University of California at San Diego (UCSD) Cancer Center, 115 patients with advanced intra-abdominal malignant conditions have been treated with several intraperitoneal chemotherapy programs. To date, with a total of 1,103 patient-months of follow-up study, seven episodes of small intestinal obstruction have developed of which two were definitely and five possibly related to chemotherapy induced adhesion formation. The development of methods to prevent or decrease peritoneal sclerosis will allow for greater application of this innovative therapeutic approach.

Adult↗
PubMed1985

High-dose intracavitary cisplatin with intravenous thiosulfate. Low incidence of serious neurotoxicity.

Recent published reports have suggested that cisplatin administered in high doses or in certain combination chemotherapy can cause serious neurotoxicity in a large percentage of patients treated. In several high-dose cisplatin-based intracavitary chemotherapy trials with the simultaneous intravenous administration of sodium thiosulfate, the incidence of clinically relevant neurotoxicity has been extremely low. In addition, several patients with serious preexisting cisplatin-induced neurologic dysfunction were treated without worsening of their clinical condition. It is suggested that thiosulfate might have been responsible for the low incidence of neurotoxicity in this patient population. Further experimental and clinical investigation of the potential of this agent to protect against cisplatin-induced neuropathy appears warranted.

Adolescent↗
PubMed1985

Cisplatin and cytarabine administered intrapleurally as treatment of malignant pleural effusions.

Eight patients with histologically-documented malignant pleural effusions received a total of ten courses of intrapleurally administered chemotherapy with cisplatin (100 mg/m2) and cytarabine (10(-2) M). Sodium thiosulfate was simultaneously administered intravenously to protect against cisplatin-induced nephrotoxicity. There was no local toxicity observed and the only significant systemic toxicity (bone marrow depression) developed in a patient with poor marrow reserve prior to the initiation of therapy. Six of seven evaluable patients exhibited major reductions (greater than 75%) in the size of their effusions lasting for 2 to 10 plus months (median: 4 months). We conclude that the intrapleural administration of this chemotherapy regimen results in objective and subjective improvement in patients with malignant pleural effusions with minimal local and systemic toxicity (except for cisplatin-induced emesis) and does not require chest tube drainage or prolonged hospitalization.

Adult↗
PubMed1985

Nephrotoxicity of high-dose intracavitary cisplatin with intravenous thiosulfate protection.

Sodium thiosulfate has been shown experimentally to protect against cisplatin-induced renal insufficiency by inactivating the nephrotoxic as well as cytotoxic properties of the agent. However, significant plasma levels of 'active' cisplatin have been demonstrated following high-dose intracavitary cisplatin administration with simultaneous intravenous thiosulfate delivery. At the UCSD Cancer Center 131 patients have been treated with a total of 485 courses (median per patient, 3; range 1-18) of intrapleural or intraperitoneal cisplatin with intravenous thiosulfate protection. Seventy-six patients (58%) had previously been treated with intravenous cisplatin. A total of 14 courses (2.9%) of intracavitary therapy were complicated by a serum creatinine rise to greater than 1.5 mg% which, in all but three cases, returned to the normal range within 1 month following treatment. All but one patient demonstrating clinical evidence of nephrotoxicity had been heavily pretreated with cisplatin. We conclude that thiosulfate can protect against clinically significant cisplatin-induced nephrotoxicity by cisplatin delivered in high doses via the intracavitary route.

Acute Kidney Injury↗
PubMed1985

Intraperitoneal chemotherapy with high-dose cisplatin and cytosine arabinoside for refractory ovarian carcinoma and other malignancies principally involving the peritoneal cavity.

Sixty-two patients with refractory ovarian carcinoma or other malignancies principally confined to the peritoneal cavity were treated with an intraperitoneal combination chemotherapy regimen consisting of cisplatin (100 mg/m2 or 200 mg/m2) and cytosine arabinoside (4 X 10(-3) mol/L or 10(-2) mol/L). Sodium thiosulfate was simultaneously administered intravenously (IV) to protect against cisplatin-induced nephrotoxicity. Sixteen of 52 evaluable patients demonstrated evidence of a clinical response including 14 (36%) of 39 with refractory ovarian carcinoma. Systemic toxicity was not severe except for cisplatin-induced emesis and a single episode of major renal insufficiency. Dose-limiting toxicity was bone marrow suppression with cytosine arabinoside administered at 10(-2) mol/L. We conclude that combination intraperitoneal therapy with high-dose cisplatin and cytosine arbinoside can be safely administered with objective tumor responses observed in patients with ovarian carcinoma refractory to front-line chemotherapy and in occassional individuals with other malignancies principally confined to the peritoneal cavity.

Adult↗