When the drug trial fails: an approach to clinical drug studies.

Drug development involves the chemical identification and characterization of a compound to determine stability and define the drug's preliminary actions. Preclinical research follows in animals to develop a pharmacokinetic profile to determine dose range, biotransformation, elimination, and toxicology. The 4 phases of clinical research, phase 1 to phase 4, encompass a progressive investigation of healthy subjects, otherwise healthy patients, to patients with a target disease to obtain US Food and Drug Administration (FDA) approval. Clinical studies include open-label noncomparative studies during phases 1 and 2, and double-blind, comparative, and placebo-controlled studies during phases 2 and 3. Approval from the FDA follows the successful evaluation of the drug. After drug marketing, phase 4 clinical trials continue to collect safety and efficacy information. Many drugs that undergo this drug development process succeed in obtaining FDA approval and are marketed for clinical use. There are several circumstances, however, that preclude the successful completion of drug development, FDA approval, and marketing. This study describes a clinical trial of a new benzodiazepine, Ro 48-6791. Ro 48-6791 was being developed as an ultra-short-acting benzodiazepine with clinical effects of shorter duration than midazolam. The purpose of this study was to define a safe dose range for the induction and maintenance of conscious sedation of patients in an outpatient gastroenterology laboratory. Efficacy criteria to be evaluated included time to onset of action, duration of action, and psychomotor fitness upon recovery. Patients were assessed by using the Observer's Assessment of Alertness/Sedation score (OAA/S) and a 5-m heel-toe-line-walk test (HTLW). The patients were divided into 2 groups. Group 1 patients received Ro 48-6791. Group 2 patients were premedicated with meperidine before administration of Ro 48-6791. Ro 48-6791 was titrated over 30 seconds, and patients were observed for 90 seconds before the next dose was given. The OAA/S score, oxygen saturation, and vital signs were charted every minute through induction and every 5 minutes during the procedure. Patients received Ro 48-6791 until they reached on OAA/S score of 3, corresponding to slowed patient response to name calling. Group 1 (Ro 48-6791 alone) required greater induction doses and increased time to induction. Maintenance doses were the same for both groups. The duration of action of Ro 48-6791 as measured by the OAA/S score and HTLW test did not differ between groups. Ro 48-6791 seemed to be a safe and effective agent to achieve conscious sedation in outpatients undergoing short invasive procedures. However, clinical drug development of Ro 48-6791 was stopped because it did not meet the efficacy criteria of an ultra-short-acting benzodiazepine.

Preoperative and postoperative nipple-areola sensibility in patients undergoing reduction mammaplasty.

Objective data on nipple and areola sensibility are scarce. For women with macromastia, there is little published information available indicating the incidence and intensity of postoperative nipple and areola sensibility. This prospective study was undertaken to evaluate nipple and areola sensibility in "small-breasted" control subjects as well as in patients with macromastia before and after reduction mammaplasty. Preoperative and postoperative Semmes-Weinstein pressure threshold testing was performed on 84 breasts in 43 patients and on 12 breasts of A or B cup size in the control group. The patients underwent reduction mammaplasty by the central parenchymal pedicle technique or the laterally based inferior pedicle technique. Nipple-areola sensibility was retained in 96 percent of breasts when the excision of breast tissue was less than 550 gm and 85 percent of breasts when the excision was greater than 550 gm. Overall, nipple-areola sensibility was retained in 90.5 percent of the 84 breasts tested. In those breasts in which nipple-areola sensibility was retained after surgery, there was no statistical difference in the preoperative and postoperative Semmes-Weinstein pressure threshold values. When pressure threshold values were compared in patients who had less than 550 gm of tissue resected, patients who had greater than 550 gm of tissue resected, and controls who had not undergone surgery, the trend of decreasing nipple-areola sensibility with increasing breast size was clearly seen.

Pharmacology of the nitrovasodilators. Antianginal, antihypertensive, and antiplatelet actions.

The nitrovasodilators, nitroglycerin and sodium nitroprusside, cause both arterial and venous smooth muscle dilation by the intracellular release of nitric oxide. Nitric oxide activates guanylate cyclase, resulting in an accumulation of cyclic GMP. The endogenous formation of nitric oxide results in vasodilatory activity similar to the nitrovasodilators. Nitroglycerin is commonly used in the treatment of angina pectoris because of its ability to decrease myocardial oxygen consumption. Most likely, this response occurs as a result of a reduction in preload, which can decrease arterial wall tension and improve coronary blood flow. This pharmacologic effect warrants the use of nitroglycerin in the treatment of myocardial ischemia or infarction, congestive heart failure, and hypertension. Sodium nitroprusside is effective in reducing arterial blood pressure in hypertensive crisis as a result of systemic vasodilation leading to a reduction in preload and afterload. Sodium nitroprusside is not as effective in the treatment of angina pectoris or in diminishing of myocardial ischemia because it does not preferentially improve blood flow to ischemic myocardium over nonischemic myocardium. Inhibition of platelet aggregation has been demonstrated with these drugs, but the clinical applications need further investigation. Nursing interventions for the patient on nitrovasodilator therapy include careful hemodynamic monitoring and drug infusion, along with elimination of physical and emotional stimuli that can aggravate the patient's underlying pathology.

Shear stress-induced release of nitric oxide from endothelial cells grown on beads.

An in vitro bioassay system was developed to study endothelium-mediated, shear stress-induced, or flow-dependent generation of endothelium-derived relaxing factor (EDRF). Monolayers of aortic endothelial cells were grown on a rigid and large surface area of microcarrier beads and were packed in a small column perfused with Krebs bicarbonate solution. The perfusate was allowed to superfuse three endothelium-denuded target pulmonary arterial strips arranged in a cascade. Fluid shear stress caused a flow-dependent release of EDRF from the endothelial cells. The action of EDRF was abolished by oxyhemoglobin and methylene blue, and the generation of EDRF in response to shear stress was markedly inhibited or abolished by NG-nitro-L-arginine, by NG-amino-L-arginine, by calcium-free extracellular medium, and by depleting endothelial cells of endogenous L-arginine. Addition of L-arginine to arginine-deficient but not arginine-containing endothelial cells rapidly restored the capacity of shear stress and bradykinin to generate EDRF. These observations indicate that fluid shear stress causes the generation of EDRF with properties of nitric oxide from aortic endothelial cells and that the bioassay system described may be useful for studying the mechanism of mechanochemical coupling that leads to nitric oxide generation.

Characterization and actions of human umbilical endothelium derived relaxing factor.

1. A bioassay cascade superfusion technique was utilized to study the properties of endothelium derived relaxing factor (EDRF) from human umbilical vein (HUV) and compare its actions on umbilical, chorionic plate and bovine pulmonary arterial strips. 2. Histamine (1 microM), bradykinin (1 microM) and A-23187 (0.3 microM, 1 microM) but not acetylcholine (1 microM) released EDRF. 3. The non-innervated human foetoplacental vessels, i.e., umbilical and chorionic plate arteries, do relax to EDRF by a guanosine 3':5'-cyclic monophosphate (cyclic GMP)-mediated mechanism. 4. The sensitivity of the human umbilical arterial strips to EDRF was less than that of the chronic plate arterial strips. Bovine pulmonary arterial strips were the most sensitive to the relaxant actions of human umbilical EDRF.

Transplantation of vascularized composite mandibular allografts in young cynomolgus monkeys.

In the search for donor tissue for massive craniofacial defects, the transplantation of somatic tissue allografts was explored. Four young, out-bred cynomolgus monkeys were the recipients of orthotopic hemimandibular allotransplants from nonrelated cynomolgus monkeys. The transplant consisted of one-half of the mandible with attached muscle, skin, and mucosa. Cyclosporine 15 mg/kg/day was given subcutaneously each day. The 4 monkeys were observed for 13, 27, 63, and 65 days, respectively. All transplants showed primary wound healing and hair growth. The 2 longest survivors chewed, ate a normal diet, and gained weight. Two allografts showed severe rejection signs at 2 to 3 weeks, and the monkeys were euthanized. One monkey had a second episode of rejection that could not be reversed, and it was killed. The fourth monkey died of undetermined causes.

Growth of vascularized composite mandibular allografts in young rabbits.

Forty-one hemimandible allografts were transplanted in young rabbits immunosuppressed with cyclosporine. The majority of the grafts demonstrated normal wound healing, and growth of hair, bone, and teeth. The mandibular body and the premolars showed significant growth in length. The allografted mandibles functioned sufficiently that the rabbits took oral nourishment soon after surgery. Long-term survival was limited by a toxic "wasting syndrome" specific for rabbits under treatment with cyclosporine.

L-arginine-dependent vascular smooth muscle relaxation and cGMP formation.

The objective of this study was to ascertain whether endothelium-dependent relaxation and guanosine 3',5'-cyclic monophosphate (cGMP) formation in bovine pulmonary artery are dependent on L-arginine. Arterial rings responded to acetylcholine and A23187 with increased cGMP accumulation and relaxation and showed resting L-arginine levels of approximately 300 microM. Addition of L-arginine failed to cause relaxation or cGMP accumulation. Arterial rings incubated under tension at 37 degree C for 24 h showed a three- to fourfold decline in L-arginine levels, and this decline was accompanied by a similar decline in resting cGMP levels as well as complete refractoriness to endothelium-dependent relaxation and cGMP formation in response to acetylcholine and A23187, without alteration of responsiveness to nitric oxide, s-nitrosothiols, or nitroglycerin. The endothelium in 24-h incubated arterial rings was normal morphologically, as assessed by scanning electron microscopy. L-Arginine caused endothelial-dependent relaxation and cGMP formation in L-arginine-depleted rings, which was antagonized by oxyhemoglobin and methylene blue. Bovine aortic endothelial cells grown in L-arginine-deficient medium supplemented with D-arginine during the final 24 h of growth failed to generate endothelium-derived nitric oxide, as assessed by bioassay cascade. L-Canavanine, but not L-lysine or L-ornithine, protected against the decline in L-arginine and cGMP levels and loss of endothelium-dependent relaxation that was characteristic of 24-h incubated arterial rings. The pharmacological properties of L-arginine were shared by L-arginine ethyl ester, L-arginine methyl ester, and L-homoarginine but not N-alpha-benzoyl-L-arginine ethyl ester or L-canavanine. These observations indicate that L-arginine or a structural analogue may be obligatory for endothelium-dependent relaxation and cGMP formation.

NG-methyl-L-arginine causes endothelium-dependent contraction and inhibition of cyclic GMP formation in artery and vein.

The objective of this study was to determine whether the vascular smooth muscle contractile effect of NG-methyl-L-arginine (NMA) is endothelium dependent and attributed to a decline in smooth muscle levels of cyclic GMP. Vascular smooth muscle levels of cyclic GMP are severalfold greater in endothelium-intact than in endothelium-denuded preparations because of the continuous formation and release of a lipophilic endothelium-derived chemical factor that diffuses into the underlying smooth muscle and activates cytosolic guanylate cyclase. This chemical substance, believed to be nitric oxide (NO) or a labile nitroso precursor, appears to account for the biological actions of endothelium-derived relaxing factor. NMA inhibits the formation of NO from endogenous L-arginine in endothelial cells. In the present study, NMA caused marked endothelium-dependent contraction of isolated rings of bovine pulmonary artery and vein, and this was similar to the contraction elicited by hemoglobin, an inhibitor of the relaxant action of NO. Both NMA and hemoglobin caused endothelium-dependent potentiation of contractile responses to phenylephrine in artery and vein. NMA caused endothelium-dependent decreases in the resting or basal levels of cyclic GMP in artery and vein to levels that were characteristic of those in endothelium-denuded vessels. Finally, NMA inhibited endothelium-dependent relaxant responses and cyclic GMP formation stimulated by acetylcholine and bradykinin. These observations reveal that interference with the continuous or basal generation of endothelium-derived NO in artery and vein can cause marked increases in vascular smooth muscle tone as a result of inhibition of cyclic GMP formation.

Antagonistic modulatory roles of magnesium and calcium on release of endothelium-derived relaxing factor and smooth muscle tone.

The objective of this study was to elucidate the mechanisms associated with the reciprocal relation between magnesium and calcium on vascular smooth muscle tone in bovine pulmonary artery and vein. Rapid removal of magnesium from Krebs-bicarbonate medium used to bathe isolated rings of precontracted artery or vein caused transient endothelium- and calcium-dependent relaxation and cyclic GMP accumulation. Both responses were antagonized by oxyhemoglobin, methylene blue, or superoxide anion and were enhanced by superoxide dismutase. The transient relaxation was followed by sustained endothelium-independent contraction. Endothelium-denuded vascular rings contracted in response to extracellular magnesium depletion without alteration in cyclic GMP levels. The data suggest that vascular endothelium-derived nitric oxide is responsible for the calcium-dependent relaxation elicited by extracellular magnesium depletion. Indeed, in bioassay cascade studies, magnesium removal from the medium used to perfuse intact artery or vein enhanced the formation and/or release of an endothelium-derived relaxing factor by calcium-dependent mechanisms. In the absence of both extracellular magnesium and calcium, calcium readdition caused transient endothelium-dependent relaxation and cyclic GMP accumulation, and both responses were abolished by oxyhemoglobin or methylene blue. In the presence of magnesium, however, readdition of calcium to calcium-depleted medium caused only contractile responses. Addition of magnesium to calcium-containing medium consistently caused endothelium- and cyclic GMP-independent relaxation that was not altered by oxyhemoglobin or methylene blue. Thus, magnesium and calcium elicit reciprocal or mutually antagonistic effects at the levels of both endothelium-derived relaxing factor formation and/or release and smooth muscle contraction. This relation may be of physiological importance, and the possibility that a reduction in circulating magnesium levels could lead to calcium-mediated vasospasm may be of pathophysiological concern.

Depletion of arterial L-arginine causes reversible tolerance to endothelium-dependent relaxation.

This study examined the influence of lowered arterial levels of L-arginine on endothelium-dependent relaxation of isolated rings of bovine pulmonary artery. Incubation of arterial rings under tension for 24 hr in oxygenated Krebs bicarbonate solution at 37 degrees C resulted in the development of marked or complete tolerance to A23187 (calcium ionophore)- and acetylcholine-elicited relaxation. Relaxant responses to nitric oxide were unaffected. Addition of L-arginine did not relax control rings but did elicit marked endothelium-dependent relaxation of tolerant rings that was inhibited by oxyhemoglobin or methylene blue. L-Arginine also restored acetylcholine-elicited relaxation. Inclusion of L-canavanine in the 24 hr incubations protected against the development of tolerance. The tissue concentration of arginine was 3-fold lower in tolerant than control arterial rings and L-canavanine restored arterial arginine levels to control values. Therefore, depletion of arterial L-arginine causes reversible tolerance to endothelium-dependent relaxation.

L-arginine causes whereas L-argininosuccinic acid inhibits endothelium-dependent vascular smooth muscle relaxation.

This study examined the actions of L-arginine, a putative precursor of endothelium-derived nitric oxide, and arginine analogs on endothelium-dependent relaxation of isolated rings of bovine pulmonary artery. L-Arginine did not consistently relax arterial rings unless rings were first rendered refractory to endothelium-dependent relaxation by pretreatment with 1 microM A23187 for 45 min. L-Arginine-elicited relaxation was endothelium-dependent, antagonized by oxyhemoglobin or methylene blue, and unaffected by indomethacin. L-Argininosuccinic acid caused endothelium-dependent contractions and irreversible inhibition of endothelium-dependent but not nitroglycerin-elicited relaxation, which was not overcome by addition of L-arginine. Inhibition of endothelium-dependent relaxation by L-NG-monomethyl arginine, however, was reversible and overcome by L-arginine. Therefore, endothelium-dependent relaxants may cause arginine depletion in endothelial cells and endogenous argininosuccinic acid may modulate the biosynthesis of endothelium-derived nitric oxide from arginine.

Poor interrater reliability of MECTA EEG recordings of ECT seizure duration.

The electroencephalogram (EEG) tracings of seizure durations recorded by the MECTA SR-1 for 24 patients undergoing right unilateral electroconvulsive therapy (ECT) were estimated independently by four psychiatrists and a neurologist. Differences among raters were often large and, overall, were statistically significant. The literature on interrater reliability for the MECTA EEG remains contradictory, and caution in MECTA EEG use appears warranted.

Growth of forelimb allografts in young rabbits immunosuppressed with cyclosporine.

Seventeen forelimbs were transplanted orthotopically from young Dutch rabbits to young New Zealand rabbits treated with cyclosporine. The transplanted limbs demonstrated significant bone growth. The growth in the transplanted limbs was about 75 to 80% of that observed in the unoperated limb. The long bones of the 3 longest surviving rabbits (133 days, 150 days, 150 days) studied radiographically demonstrated increases in length over their original lengths (humerus 22%, ulna 26%, and radius 31%). Hair and nail growth were noted at about day 10. Response to pain stimuli (withdrawal of forelimb) and functional use (ambulation with 50% weight bearing) was seen at two to three months. Permanent survival was not achieved because of a species-specific toxic wasting syndrome from cyclosporine.

Basic polyamino acids rich in arginine, lysine, or ornithine cause both enhancement of and refractoriness to formation of endothelium-derived nitric oxide in pulmonary artery and vein.

The objective of this study was to elucidate the mechanism by which polyamino acids containing L-arginine, L-lysine or L-ornithine cause endothelium-dependent relaxation of bovine intrapulmonary artery and vein. Basic but not acidic or neutral polypeptides ranging in average molecular weights from 17 to 225 kDa elicited time- and concentration-dependent relaxation and cyclic GMP accumulation in precontracted rings of artery and vein by endothelium-dependent mechanisms. Vascular responses were markedly inhibited by oxyhemoglobin, methylene blue, or potassium. The basic polyamino acids stimulated the formation and/or release of an endothelium-derived relaxing factor (EDRF) identified as nitric oxide (NO) in perfused segments of both artery and vein as assessed by bioassay. The polyamino acids and A23187 released a similar endothelium-derived NO (EDNO) from artery and vein, as assessed by the similar half-life (3-5 seconds), antagonism by superoxide anion or oxyhemoglobin, enhancement by superoxide dismutase, and lack of influence by indomethacin. The basic polyamino acids elicited potent relaxant responses with EC50 values ranging from 3 x 10(-9) to 2 x 10(-7) M, and a direct correlation was obtained between molecular weight and relaxation potency irrespective of the basic amino acid incorporated. Prolonged contact of arterial or venous rings with basic polyamino acids resulted in the rapid development of marked refractoriness to relaxation and cyclic GMP formation on addition of polyamino acid. Moreover, refractoriness developed to the vascular responses of other endothelium-dependent vasodilators but not to glyceryl trinitrate or isoproterenol. The mechanism of refractory responses was attributed to interference with EDNO formation and release as assessed by bioassay and chemical assay. The hypothesis is forwarded that the basic polyamino acids serve as partial substrates for the enzyme system that catalyzes the conversion of L-arginine to NO. Prolonged contact between substrate and enzyme results in enzyme desensitization and the development of refractoriness or a form of tolerance to vasodilators whose action is mediated by EDNO.

Endothelium-derived nitric oxide relaxes nonvascular smooth muscle.

A bioassay cascade superfusion procedure was used to compare and contrast the actions of arterial and venous endothelium-derived relaxing factor (EDRF) with authentic nitric oxide (NO) on several nonvascular smooth muscle preparations. EDRF was released from human umbilical vein or bovine pulmonary artery by A23187 and allowed to superfuse two nonvascular and one vascular precontracted smooth muscle strips arranged in a cascade. NO or S-nitroso-N-acetylpenicillamine was delivered by superfusion. Both EDRF and NO relaxed bovine trachea, although artery was 10 times more sensitive than trachea to either relaxant. Similarly, rabbit taenia coli and rat fundus relaxed in response to high concentrations of NO or large amounts of EDRF released from umbilical vein. Vascular and nonvascular relaxant responses to both EDRF and NO were inhibited by oxyhemoglobin, methylene blue or superoxide, and were enhanced by superoxide dismutase. Perfusion of pulmonary artery or umbilical vein with A23187 resulted in contraction of guinea pig ileum and relaxation of pulmonary artery, whereas NO relaxed both preparations. Oxyhemoglobin enhanced the contractile and abolished the relaxant responses. Thus, ileum is more sensitive to endothelium-derived contracting factor(s) than to EDRF. NO raised cyclic GMP levels in all smooth muscle preparations, but a greater fold increase was observed in artery than in nonvascular smooth muscle. EDRF released from human umbilical vein was identified chemically as NO or a nitroso compound, as was done previously for EDRF from bovine pulmonary artery and vein. These observations support the view that one EDRF from artery and vein is NO or a labile nitroso compound.

Reconstruction of axillary burn contractures with the latissimus dorsi fasciocutaneous flap.

Standard management of axillary burn contractures has been scar release and the use of skin grafts, despite the common problem of incomplete graft take, prolonged splinting, extended physical therapy, and recurrent contractures. A recent development in plastic surgery has been the "super flap" or fasciocutaneous flap. A series of axillary burn contractures released with the latissimus dorsi fasciocutaneous flap has been reported by Tolhurst. Our series of ten patients confirms that the latissimus dorsi fasciocutaneous flap is the treatment of choice for the release of severe axillary burn contractures.

Factors influencing vascular hyporesponsiveness to angiotensin II.

Bartter's syndrome is characterized, in part, by hyporesponsiveness to the pressor effect of exogenous angiotensin II (AII). This has been attributed to volume contraction, hypokalemia, and/or increased prostaglandin (PG) levels. In order to investigate factors responsible for a diminished response to the pressor effect of AII, rats were made hypokalemic or volume contracted and hypokalemic (VCHK) by dietary restriction. AII sensitivity was examined by determining the dose of AII required to raise the mean arterial pressure 20 mm Hg. When compared with control rats. VCHK and hypokalemic rats were significantly less sensitive to AII. VCHK rats were significantly less sensitive to AII than hypokalemic rats. Both experimental groups were similarly hypokalemic, but plasma renin activity (PRA) of VCHK only was greater than control values. In VCHK rats, acute K+ restoration partially corrected AII hyporesponsiveness, although plasma K+ increased to normal. In VCHK rats, acute volume expansion with normal saline similarly achieved only partial correction of AII hyporesponsiveness although PRA values fell to the control range. Simultaneous K+ restoration and volume expansion to VCHK rats successfully restored AII sensitivity to the control range. Dietary sodium, chloride, and potassium restriction did not increase urinary excretion to PGE2. Indomethacin (5 mg/kg, iv) given acutely to VCHK rats did not significantly after baseline hyporesponsiveness to AII. Norepinephrine vascular sensitivity was not affected by either volume contraction or hypokalemia. These data demonstrate that volume contraction and hypokalemia individually depress exogenous AII sensitivity in the rat and do so by separate and additive mechanisms. Furthermore, these mechanisms appear to be independent of PG.