Utility of clinical high-depth next generation sequencing for somatic variant detection in the PIK3CA-related overgrowth spectrum.

Next-generation sequencing (NGS) has revolutionized the approach of studying sequence variation, and has been well described in the clinical laboratory setting for the detection of constitutional alterations, as well as somatic tumor-associated variants. It is increasingly recognized that post-zygotic somatic alteration can be associated with congenital phenotypic abnormalities. Variation within the PI3K/AKT/mTOR pathway, including PIK3CA, has been described in somatic overgrowth syndromes and vascular malformations. Detection of PIK3CA somatic alteration is challenging because of low variant allele frequency (VAF) along with the need to assay involved tissue, thus necessitating a highly sensitive methodology. Here we describe the utility of target hybrid capture coupled with NGS for the identification of somatic variation in the PIK3CA-related overgrowth spectrum (PROS) among 14 patients submitted for clinical testing. Assay detection of low allelic fraction variation is coverage dependent with >90% sensitivity at 400× unique read depth for VAF of 10%, and approaching 100% at 1000×. Average read depth among the patient dataset across PIK3CA coding regions was 788.4. The diagnostic yield among this cohort was 71%, including the detection of two PIK3CA alterations novel in the setting of PROS. This report expands the mutational scope and phenotypic attributes of PROS disorders.

Single versus triplicate measurements of blood pressure and heart rate.

The mean of rapidly repeated duplicate or triplicate measurements is often used in studies of antihypertensive drugs. Forty patients with hypertension had triplicate measurements of blood pressure and heart rate on two occasions, 1 week apart, during placebo treatment. The average difference between the first measurement and the mean of the triplicate measurements was -0.3 mm Hg. The average coefficient of variation for supine and standing, systolic and diastolic blood pressures was 8.4% for the single measurements and 8.0% for the mean of triplicate measurements. The correlations between the first measurements and the mean of triplicate measurements ranged from 0.90 to 0.98 (all p less than 0.01). The average difference between the two visits for all four blood pressure parameters was -0.6 mm Hg for the single measurements and -0.5 mm Hg for the mean of triplicate measurements (all p = NS). These results indicate that 1) blood pressure does not change further after 1 week of placebo treatment, and 2) use of the mean of triplicate measurements of blood pressure and heart rate gives the same result as use of single measurements, and the results are no less variable.

Clodronate kinetics and dynamics.

Clodronate disodium (Cl2MDP) was given intravenously in doses of 3, 6 and 10 mg/kg to six men (aged 23 to 30 yr). Volume of distribution was 0.2720 +/- 0.0255 l/kg (mean +/- SD) after 3 mg/kg dose, 0.3037 +/- 0.0445 l/kg after 6 mg/kg, and 0.2528 +/- 0.417 l/kg after 10 mg/kg. The elimination rate constant was 0.3787 +/- 0.0546 hr(-1), 0.3492 +/- 0.0616 hr(-1), adn 0.3962 +/- 0.0358 hr(-1) after 3, 6, and 10 mg/kg. Corresponding total body clearances were 0.1026 /+- 0.0149, 0.1049 +/- 0.0159, and 0.0998 +/- 0.0172 l/kg/hr. Renal clearance accounted for 73% of total body clearance; 73% of the drug was extracted unchanged in the urine in 24 hr. After Cl2MDP serum phosphate decreased approximately 13%; this was associated at the 10 mg/kg dose with a transient fall in fractional phosphate excretion. There was no significant changes in the serum concentration of fractional excretion of calcium, sodium, or uric acid. Creatinine clearance and renal concentrating ability were not altered by Cl2MDP. After short-term administration Cl2MDP is excreted primarily by the kidney but has no significant effects on renal function.

Effects of metoprolol and propranolol on theophylline elimination.

The effects of beta adrenergic blockade on theophylline elimination were studied in nine nofmal subjects. Oral propranolol, 40 mg every 6 hr, induced a fall in theophylline clearance from 0.0464 +/- 0.0294 +/- 0.0129 l/kg/hr (p < 0.001). Oral metoprolol, 50 mg every 6 hr, did not reduce theophylline clearance in the group as a whole but had reducing effect intermediate to that of propranolol on theophylline clearance in some smokers whose theophylline clearance was high initially. Beta adrenergic blockade may reduce theophylline clearance, particularly in subjects whose theophylline metabolism has been induced by cigarette smoking.

Gastrointestinal absorption as a function of age: xylose absorption in healthy adults.

Xylose oral absorption was examined in 24 healthy male subjects ranging in age from 32 to 85 years. Absorption was evaluated from xylose plasma concentration-time data after administration of a 25 gm po or a 5 gm iv dose. There was no relationship between various estimates of the rate of absorption and age. The absolute oral bioavailability or the extent of xylose absorption showed no relationship to age in our population. In contrast with previous suggestions, xylose absorption does not decline with age. General statements of decreased gastrointestinal absorption efficiency as a function of age may not be correct.

Xylose disposition in humans as a function of age.

D-xylose disposition was examined in 24 healthy men between 32 and 85 years of age. Xylose was administered as a 5 gm iv infusion and as a 25 gm po solution. Serum xylose concentrations and urinary excretion of intact xylose were determined. There were statistically significant inverse relationships with age for each of the following parameters after intravenous infusion: elimination rate constant (r2 = 0.71); systemic clearance (r2 = 0.66); renal clearance (r2 = 0.66); and nonrenal clearance (r2 = 0.35). Similar inverse relationships were found after oral dosing for the elimination rate constant (r2 = 0.69) and renal clearance (r2 = 0.54). There was no significant age relationship for the apparent volume of distribution or the steady-state volume of distribution. The percentage of the oral and intravenous dose recovered in urine up to 5 hours after dosing was significantly and inversely correlated with age. The implications of the latter finding are discussed with regard to the interpretation of the xylose tolerance test used to assess gastrointestinal absorptive capacity.

Effects of meals on hemodynamics: implications for antihypertensive drug studies.

The ingestion of food is known to affect blood pressure and heart rate, but food is often allowed in patients under observation for antihypertensive drug effects. Seventy-seven patients with essential hypertension were observed for 8 hours after a 16-hour fast. Thirty-six continued to fast, 20 ate a high-carbohydrate meal, and 21 ate a meal of their own choice. Blood pressure and heart rate did not change during fasting, but both meals lowered mean supine and standing diastolic blood pressures during the subsequent 4 hours by 3 to 7 mm Hg (P less than 0.001). The high-carbohydrate meal reduced supine systolic blood pressure by 6 mm Hg (P less than 0.0001). Both meals increased supine and standing heart rates by 5 to 8 bpm (P less than 0.001). After the self-selected meal, standing systolic blood pressure increased in younger patients but decreased in older patients. Food ingestion during antihypertensive drug studies may interfere with the interpretation of results and should be avoided whenever possible.

Lidocaine elimination: effects of metoprolol and of propranolol.

The effects of administration of metoprolol and propranolol on lidocaine elimination were studied in six healthy young men who did not smoke. Each received three single intravenous doses of lidocaine (2.5 to 3.0 mg/kg injected over 10 min): one alone, one after 1 day pretreatment with propranolol (40 mg orally every 6 hr), and one after 1 day pretreatment with metoprolol (50 mg orally every 6 hr). Lidocaine clearance was 0.88 +/- 0.28 l X hr-1 X kg-1 before beta blockade, 0.61 +/- 0.20 l X hr-1 X kg-1 during metoprolol dosing, and 0.47 +/- 0.16 l X hr-1 X kg-1 during propranolol dosing. There was no correlation between the change in lidocaine elimination and the steady-state concentrations of metoprolol or propranolol, nor between the change in lidocaine clearance and the change in resting heart rate produced by either beta blocker. Metoprolol and propranolol reduce lidocaine elimination significantly.

Effects of ketorolac tromethamine on hemostasis in volunteers.

Ketorolac tromethamine, an analgesic agent with prostaglandin synthetase--inhibiting activity, is more active than aspirin in vitro in inhibiting collagen- or arachidonic acid-induced platelet aggregation. In this randomized, double-blind study, 26 volunteers received ketorolac, 30 mg intramuscularly four times a day for 5 days, and placebo, two capsules orally four times a day for at the last 2 study days. The effects of this treatment were compared with those of intramuscular placebo and oral aspirin, two 325 mg capsules, given on the same schedule to eight volunteers. Aspirin at a mean serum concentration of 84 micrograms/ml did not affect prothrombin time, partial thromboplastin time, platelet count, or bleeding time. Ketorolac produced a modest prolongation of the bleeding time, from 4.9 +/- 1.1 minutes (mean +/- SD) to 7.8 +/- 4.0 minutes (p less than 0.005). Ketorolac did not affect the prothrombin time or partial thromboplastin time but was associated with clinically insignificant change in the platelet count from 303 +/- 57 X 10(3)/m3 to 277 +/- 56 X 10(3)/mm3.

Antihypertensive and biochemical dose-response study of tripamide.

Tripamide is an experimental sulfonamide-derived diuretic antihypertensive agent. Twenty-four hospitalized patients with essential hypertension received placebo followed by 10, 25, 50, or 100 mg of tripamide daily in a randomized, double-blind design. All doses of tripamide significantly lowered standing arterial pressure. Changes in blood pressure, heart rate, and weight were not dose related, but the decrease in mean arterial pressure was significantly related to both age (P less than 0.02) and pretreatment blood pressure (P less than 0.05). Serum potassium levels were lowered significantly by the 25 and 100 mg daily doses of tripamide, whereas all doses of tripamide significantly reduced serum chloride levels and produced an increase in serum uric acid levels. Disparate time courses of antihypertensive and diuretic effects and the lack of a relationship between the increase in urine volume and the change in blood pressure suggest an additional antihypertensive action of tripamide or a delayed physiologic adaptation to volume depletion. Equal antihypertensive effects over the range of 10 to 100 mg/day, but greater hypokalemia at higher doses, suggest that future studies should employ the lower doses of tripamide.

Effect of tripamide on glucose tolerance in patients with hypertension.

The effects of tripamide and hydrochlorothiazide on blood pressure and glucose tolerance were studied in 20 hypertensive patients, half of whom had type II diabetes mellitus. Each patient underwent intravenous glucose tolerance testing before and after 4 weeks of treatment with tripamide, 10 mg, and, at a separate time, hydrochlorothiazide, 50 mg. Both tripamide and hydrochlorothiazide lowered blood pressure; for both drugs, the magnitude of the reduction in mean arterial pressure was positively correlated with the pretreatment mean arterial pressure. Hydrochlorothiazide produced a greater fall in serum potassium than did tripamide. In the nondiabetics, neither drug produced a significant change in the glucose disappearance curve or the plasma insulin response. In the diabetics, hydrochlorothiazide produced an increase in serum glucose levels, but the plasma insulin response, which was blunted in comparison to the nondiabetics, did not change. Tripamide did not affect serum glucose or plasma insulin levels in either group of patients. Tripamide at a dose of 10 mg daily does not affect glucose tolerance in either nondiabetic hypertensive patients or patients with type II diabetes mellitus.

Antihypertensive effects of parenteral nicardipine alone and in combination with captopril.

We studied the safety and efficacy of intravenous nicardipine alone and in combination with oral captopril. Sixteen patients with essential hypertension received a single oral dose of captopril, 50 mg, to be certain that excessive hypotension would not occur. Nicardipine was given intravenously as a 2 mg bolus, followed by an infusion at a rate designed to lower the supine diastolic blood pressure at least 10 mm Hg; then oral captopril, 50 mg, or placebo was given. The next week, nicardipine was again infused, but the alternate oral treatment was given. Intravenous nicardipine reduced blood pressure from 156 +/- 15/101 +/- 5 mm Hg (mean arterial blood pressure 120 +/- 6 mm Hg) to 140 +/- 11/88 +/- 4 mm Hg (mean arterial blood pressure 105 +/- 5 mm Hg). When captopril was added to nicardipine, the mean arterial blood pressure fell an additional 8 mm Hg but the heart rate did not increase. The combination of angiotensin-converting enzyme inhibition and calcium channel blockage produces additive antihypertensive effects without additional reflex tachycardia.

Nicardipine and hydrochlorothiazide in essential hypertension.

Nicardipine is an investigational dihydropyridine calcium channel blocking agent. One hundred fifty-one patients with hypertension received either 30 mg nicardipine t.i.d. or 25 mg hydrochlorothiazide b.i.d. in a double-blind, randomized, multicenter trial. After 4 weeks of therapy and at the end of the dosing interval, nicardipine reduced arterial pressure by 10/6 mm Hg and 12/6 mm Hg in the supine and standing positions, respectively (all p less than 0.01). In the hydrochlorothiazide group, the reductions were 12/6 mm Hg and 14/6 mm Hg, respectively (all p less than 0.01). The maximum reduction in blood pressure of 16/14 mm Hg supine and 20/15 mm Hg standing occurred within 1 hour after administration of nicardipine. The mean reduction in the hydrochlorothiazide group after 1 hour was 14/11 mm Hg supine and 16/12 mm Hg standing. Neither drug affected autonomic reflexes associated with maximum exercise. Nicardipine increased urinary sodium excretion during the 4-hour period after the first dose. Adverse effects of nicardipine were primarily extensions of its vasodilator effect and included flushing, headache, and edema.

Effect of quinine on digoxin kinetics.

Six subjects were evaluated for the effect of quinine, the l-isomer of quinidine, on digoxin pharmokinetics. A 1.0-mg intravenous digoxin dose was given before and during quinine administration, followed by the measurement of digoxin serum and urine concentrations for 96 hr after each dose. Quinine reduced digoxin total body clearance by 26% from 2.98 to 2.22 ml/min/kg (p < 0.03). Digoxin elimination half-life (t 1/2) was lengthened from 34.2 to 51.8 hr, reflecting a 32% decrease in digoxin elimination rate constant (p < 0.003). Quinine did not reduce digoxin renal clearance or any volumes of distribution. The amount of digoxin excreted into the urine increased from x = 628. 29 micrograms to x = 772.52 micrograms (p < 0.02). Digoxin nonrenal clearance decreased an average of 55% from 1.2 to 0.55 ml/min/kg (p < 0.05). These results suggest that quinine alters digoxin metabolism or biliary secretion, reducing digoxin total body clearance by a mechanism that is qualitatively similar, but quamtitatively different, from quinidine.

Vitamin C disposition in young and elderly men.

The pharmacokinetics of vitamin C after a 500-mg oral tablet dose were compared in 15 healthy young men aged 25.20 +/- 2.54 (means +/- SD) and in 15 healthy elderly men aged 69.31 +/- 2.52 y. The doses were characterized with the subjects in two states of vitamin C nutriture: a depleted state, which was achieved by 4-5 wk of compliance with a vitamin C-restricted diet of less than 10 mg/d and a supplemented state, in which the subjects were given 500 mg vitamin C/d for 3 wk. Plasma and urine samples were collected for 72 h after the dose of vitamin C from depleted subjects and for 24 h from supplemented subjects and analyzed for vitamin C. Several of the pharmacokinetic indices measured were different in depleted vs supplemented subjects but none exhibited any age-related differences. This indicates that vitamin C nutriture affects vitamin C pharmacokinetics but age does not.

The effect of vitamin C on the pharmacokinetics of caffeine in elderly men.

The influence of vitamin C on the pharmacokinetics of caffeine was investigated in 10 elderly males, age 66 to 86 yr. Caffeine was administered intravenously on three different occasions over a 7-wk period: before vitamin C restriction, after approximately 4 wk of vitamin C restriction (15 mg dietary intake per day), and after 2 wk of vitamin C supplementation (500 mg orally, twice daily). Blood and urine samples were collected over a 48-h period after each caffeine administration. The plasma half-life, rate constant of elimination, apparent volume of distribution, total body clearance, renal clearance, and metabolic clearance of caffeine were determined. Simultaneous plasma, whole blood and leukocyte vitamin C concentrations were obtained. All of the average vitamin C concentrations monitored (plasma, whole blood, and leukocyte) changed significantly during the study, corresponding to the alterations in dietary vitamin C intake. Conversely, none of the caffeine pharmacokinetic parameters evaluated changed significantly during the study. The average metabolic clearance was approximately 77 (ml hr-1) kg-1 and the average half-life was approximately 4.6 h for all caffeine administrations. These results indicate that the elimination of caffeine in the elderly is not affected significantly by the concentrations of vitamin C achieved during this study.

Cardiovascular effects of caffeine in elderly men.

Ten healthy elderly male volunteers were given 4 mg/kg of caffeine by intravenous infusion on three separate occasions. This resulted in mean peak plasma concentrations of caffeine of 7.4 +/- 0.7 micrograms/ml. Immediately after each of the three caffeine infusions, the mean systolic blood pressures increased 14, 7, and 16 mm Hg, and the mean diastolic blood pressures increased 7, 4, and 7 mm Hg, respectively. Both the systolic and the diastolic blood pressures returned to preinfusion values within 4 hours. The QS2 index and the left ventricular ejection time (LVET) index increased after caffeine, probably as a result of the caffeine-induced increase in arterial blood pressure. The pre-ejection period (PEP), the PEP/LVET ratio, and the diastolic time remained unchanged. Caffeine, in doses equal to those contained in 2 to 3 cups of coffee, produces an increase in blood pressure but has no demonstrable positive inotropic effect in healthy elderly men.

Comparison of the inotropic and chronotropic effects of metoprolol and propranolol.

The cardiovascular responses to intravenous doses of isoproterenol were measured in eight male volunteers before and during administration of 50 mg metoprolol, orally every 6 hours and 40 mg propranolol, orally every 6 hours for a total of five doses. The dose of isoproterenol required to produce an increase in heart rate of 25 beats/min (the ID25) was 2.0 +/- 1.4 microgram before beta blockade, 6.2 +/- 4.4 microgram during metoprolol, and 44.4 +/- 12.0 microgram during propranolol administration. Similar changes in diastolic blood pressure, QS2I, preejection period, and preejection period/left ventricular ejection time ratio occurred at the ID25 during treatment with both metoprolol and propranolol. In volunteers, propranolol produces a much more intense blockade of the inotropic and chronotropic effects of isoproterenol than does metoprolol.

The influence of age on salicylate pharmacokinetics in humans.

The pharmacokinetics of salicylate after a single oral solution dose of 600 mg of sodium salicylate were investigated in 22 male subjects. Subjects were healthy nonsmokers and were not taking any regular medication. The plasma concentration and urinary excretion of salicylic acid and its metabolite, salicyluric acid, as well as the urinary excretion of salicyl glucuronides were determined. Urinary recovery essentially accounted for the administered dose and was not influenced by age, nor was the apparent oral clearance of salicylic acid. Assuming no presystemic elimination, it could be concluded that systemic availability is unaffected by age. An increase in the apparent volume of distribution, Varea, and a decrease in the maximum plasma salicylic acid concentration with age were observed. Renal clearance of salicyluric acid decreased significantly with age and was found to correlate significantly with creatinine clearance. The authors conclude that age does not have a major influence on salicylate disposition in healthy adult men.

The influence of hydrochlorothiazide and tripamide on serum and urinary amylase.

Pancreatitis and asymptomatic elevations of serum amylase have been reported after therapy with thiazide diuretics. In the current study, the effects of hydrochlorothiazide and tripamide treatment on serum and urinary amylase excretion were investigated in 12 hypertensive volunteers. Two patients developed modest elevations of the serum amylase above the normal range after 12 weeks of treatment with hydrochlorothiazide 50 mg daily, but the mean serum amylase did not change. Hydrochlorothiazide did not produce a statistically significant increase in urinary amylase excretion but did reduce the ratio of salivary amylase/creatinine clearance in a two-hour urine collection. Tripamide 10 mg daily had no effect on serum or urinary amylase.