Distribution and degradation of [3H]methotrexate after intravenous and cerebral intraventricular injection in primates.

Four hr after either a single injection or continuous infusion of methotrexate (MTX) plus purified [3',5',9(n)-3H]MTX in cynomolgus or rhesus monkeys, 80 to 98% of the 3H radioactivity present in the plasma was found not to represent intact MTX. The percentage of 3H-containing MTX products in the urine after 4 hr was considerably less, although more variable. This variability seemed to be related to variability in the amount of the total dose excreted. Non-MTX products were also found in selected tissues and the percentage of intact MTX found 4 hr after i.v. injection varied from 2 to 26%. The percentage of intact MTX was routinely measured by comparing the values obtained using the dihydrofolate reductase assay with values based on the specific activity of [3',5',9(n)-3H]MTX. Results obtained by diethylaminoethyl column chromatography on a few samples, however, showed good agreement with results from the reductase assay. [3',5',9(n)-3H]MTX products appeared in peaks eluting from the diethylaminoethyl column both earlier and later than the MTX peak, with the earlier peaks being present in only small amounts in the urine. After continuous i.v. infusion, only 2% or less of the radioactivity found in the cerebrospinal fluid after 4 hr represented intact MTX, with the remaining radioactivity eluting much earlier than MTX. In contrast, after direct injection into the left lateral ventricel, all the 3H radioactivity in both cerebrospinal fluid and brain tissue represented intact MTX for up to 4 hr after injection. The appearance of MTX products in the plasma and selected tissues of these primates a short time after i.v. injection is compared to other work in experimental animals and man and suggests a greater metabolism of MTX than was previously suspected.

The effect of entrapment in liposomes on the in vivo distribution of [3H]methotrexate in a primate.

Entrapment of methotrexate (MTX) plus [3',5', 9(n)-3H]methotrexate into positively charged liposomes greatly alters the subsequent distribution of [3H]MTX in a cynomologous monkey (Macaca irus) after a single i.v. injection ([3H]MTX; refers to total radioactivity derived from purified [3H]MTX). When [3H]MTX is incorporated into small, sonically disrupted liposomes, the level of the entrapped [3H]MTX in the total plasma volume was still 50% of the total injected dose after 4 hr, which is 100 times greater than the level found when the same amount of free MTX (1 to 4 mg) plus [3H]MTX was injected. When entrapped in larger mechanicanically disperesed liposomes, however, the plasma levels of liposome-entrapped [3H]MTX at 4 hr was only 6-fold greater than free [3H]MT. The liposome-entrapped MTX (refers to MTX measured by dihydrofolate reductase assay) did not show detectable breakdown in the plasma whereas free MTX showed up to 97% breakdown. Increased clearance of [3H]MTX entrapped in mechanically dispersed liposomes was complemented by its much greater uptake into tissues, especially spleen, compared with sonically disrupted liposomes. There was over a 160-fold increased uptake by the spleen of liposome-entrapped [3H] MTX relative to free [3H]MTX, whereas for sonically disrupted liposomes the comparable ratio was 20. Although this liposome-entrapped MTX showed significant breakdown, it was less than that found after injection of free MTX. In certain tissues, especially the small intestine, a reduced uptake of liposome-entrapped [3H]MTX WAS FOUND. Uptake of liposome-entrapped [3H]MTX into liposomes led to a much lower renal clearance of [3H]MTX, especially in the case of sonically disrupted liposomes. Possible reasons for these effects and the relationship of our findings to those of others are discussed.

Inhibition of renal tubular transport of methotrexate by probenecid.

The mechanism of excretion of methotrexate (MTX) has been investigated in the monkey. Under steady-state conditions of varied plasma levels of MTX, it was determined that MTX was excreted by renal tubular transport as well as by glomerular filtration. The maximum rate of renal tubular transport of MTX (81 mug/min) was attained at plasma levels of MTX from 6 to 8 mug/ml. Correspondingly, the rate of clearance of MTX from plasma was shown to diminish from a value that was 3-fold greater than the glomerular filtration rate at plasma levels of MTX from 6 to 32 mug/ml. Pretreatment of animals with probenecid (700 mg/sq m) totally inhibited renal tubular transport of MTX when MTX was administered in doses from 1.8 to 621 mg/sq m. Following inhibition of renal tubular transport of MTX by probenecid, steady-state plasma levels of MTX in animals pretreated with probenecid (700 mg/sq m) was reduced by a factor of 2.6 from values determined in non-probenecid-pretreated control animals receiving similar varied doses of MTX (1.8 P to greater than 600 mg/sq m). The mode of i.v. injection of MTX was seen to effect the concentration of MTX in plasma. Initial loading followed by continuous sustaining infusion of MTX provided stable and higher levels of MTX in plasma than was determined in controls or in experimental animals pretreated with probenecid and receiving identical doses of MTX by single bolus injection.

Agents for the treatment of brain edema. 2. [(2,3,9,9a-Tetrahydro-3-oxo-9a-substituted-1H-fluoren-7-yl)oxy]+ ++alkanoi c acids and some of their analogues.

Our initial paper discussed brain edema resulting from traumatic head injury and the need for specific and effective agents to treat the disorder and disclosed a novel approach for the discovery of a drug of this kind. The current study describes the synthesis of a series of [(2,3,9,9a-tetrahydro-3-oxo-9a-substituted-1H-fluoren-7-yl)oxy]alk anoic acids and their analogues. These compounds were evaluated in an in vitro cerebrocortical tissue slice assay for their relative potencies in inhibiting K+ + HCO3- induced swelling. Structural modification at a number of sites in the "lead" compound revealed that significant biological activity was inherent only within a very narrow range of structural types. The observation that nearly all the biological activity resided in one of the two enantiomers demonstrated the marked stereospecificity of the most active compounds. One of the most potent compounds, (R)-(+)-[(5,6-dichloro-2,3,9,9a-tetrahydro-3-oxo-9a-propyl-1H-fluoren -7-yl) oxy]acetic acid ((+)-5c), exhibited a dose-response relationship in the in vivo acceleration/deceleration brain edema assay, and the data from the two highest doses were statistically significant. Electron microscopic examination demonstrated that the perivascular astroglial swelling that arises from this procedure is abolished in the animals treated with (+)-5c. This compound is currently being evaluated for its clinical efficacy and safety in the treatment of traumatic head injury.

Agents for the treatment of brain injury. 1. (Aryloxy)alkanoic acids.

Blunt and ischemic injuries of the brain have been shown to result in swelling that is predominantly limited to a single cell type, the astrocyte, within the complex cellular mosiac of cerebral gray matter. Evaluation of various diuretic (aryloxy)acetic acids in vitro using incubating cat brain slices and primary astrocyte cultures identified compounds with marked ability to inhibit brain tissue swelling. Some of the compounds significantly reduced the mortality and morbidity following acceleration/deceleration brain injury in anesthesized cats. A variety of (indanyloxy)alkanoic acids were synthesized which were analogous to the dually active (indanyloxy)acetic acids. Some of the 4-(indanyloxy)butanoic acids were found to be devoid of diuretic activity but to possess equal or greater activity than the dually active compounds in the in vitro and in vivo brain assays. Selected examples from both the (indanyloxy)acetic and 4-(indanyloxy)butanoic acid series showed marked chiral effects, with one enantiomer generally exhibiting a much greater activity than the other. A clinical study of severely head-injured patients treated with ethacrynic acid demonstrated a significantly improved outcome when compared to controls. These data suggest a clinical advantage for the nondiuretic (aryloxy)alkanoic acids which possess in vitro and in vivo activities in the cat brain assays that are comparable or superior to dually active compounds.

SITS-inhibitable Cl- transport and Na+-dependent H+ production in primary astroglial cultures.

The uptake and efflux of Cl- were measured in primary astroglial cultures from neonatal rat brain using 36Cl- as a tracer. Both uptake and efflux were found to be inhibited by the specific anion inhibitor SITS. The rate of Cl- efflux showed a broad optimum at pH values greater than 7.5, and both this pH dependence and the effect of SITS suggests that these cells contain a Cl- in equilibrium Cl- or Cl- in equilibrium HCO3- exchange carrier similar to that described in erythrocytes. In addition, the cells rapidly lost Cl- when placed in media of decreasing Cl- concentrations, and ploting the initial rate of uptake of 36Cl- as a function of external Cl- concen-ration gave an apparent Km for Cl- uptake of 56 mM. Pretreatment of these cultures with DBcAMP is known to cause the cells to form numerous processes, resulting in their morphology more closely resembling that of astroglia in brain. Treatment with DBcAMP resulted in decreased equilibrium levels of 36Cl- and a small decrease in the initial rate of uptake of 36Cl-, but did not affect inhibition by SITS. Addition of Na+ to the cells suspended in Na+-free media specifically increased the rate of acidification of the medium. These observations suggest that these cells have both Cl- in equilibrium HCO3- and Na+ in equilibrium H+ exchange processes which, if these cultures can be considered to be representative of cells in vivo, may also occur in astroglial cells in the central nervous system. Based on these results and other work, a model is proposed by which these processes would lead to the astroglial swelling which is often observed in vivo in pathological conditions.

Enzymatic and morphological properties of primary rat brain astrocyte cultures, and enzyme development in vivo.

The development of (Na+ + K+) ATPase, carbonic anhydrase and HCO3--stimulated ATPase activity was studied in developing rat brain in vivo, and in primary astrocyte cultures from 1--3-day-old rat brain as a function of increasing cell growth. The primary cultures showed an increase in all the above enzyme activities during cell growth, with time courses which were qualitatively similar to their development in vivo. Cell cultures grown separately from the cerebellum plus brain stem regions showed greater carbonic anhydrase activity than cerebral cultures over the entire 4-week growth period, corresponding to development of this activity in these same regions in vivo, HCO3-stimulated ATPase activity was slightly greater in cerebellar cultures and (Na+ + K+) ATPase activity was greater in cerebral cultures up to the second week of growth, resembling development of the same enzyme activities in vivo. C6 glioma and neuroblastoma cells showed no and 10-fold lower carbonic anhydrase activities respectively, compared to the primary astrocyte cultures. Addition of 1 mM N6-2'-O-dibutyryladenosine-3',5'-monophosphate (DBcAMP) in the presence of serum caused marked formation of cellular processes and increased carbonic anhydrase and (Na+ + K+) ATPase activity. Maximum effects were found 2 h after addition of 1 mM DBcAMP and thereafter declined. In the absence of serum such effects persisted for at least 24 h. Electron microscope studies showed large numbers of microtubule (approximately 20 nm diameter) and filamentous structures (less than or equal to 10 nm diameter) in the cytoplasm, which showed changes in distribution in cells treated with DBcAMP. This study suggests that the increase in ATPase and carbonic anhydrase activities in rat brain with increasing age may be in part a reflection of proliferation and development of astroglia cells. Together with the morphological data, it also provides additional evidence that primary cultures derived from neonatal rats may closely resemble developing astroglia in vivo.

Cation transport and membrane potential properties of primary astroglial cultures from neonatal rat brains.

This paper describes K+ and Na+ content and transport in primary monolayer cultures from dissociated newborn rat brains, considered to consist predominantly of astroglial cells. Net changes in cation content after addition of ouabain, and steady state fluxes using 86Rb+ as a marker for K+ and 22Na+ as a marker for Na+, were measured. The results found indicate that the cells maintained a conventional pattern of cation homeostasis with net efflux of K+ being balanced by its active uptake and net uptake of Na+ balanced by active extrusion mediated by a ouabain sensitive (Na+K) pump. These processes maintained internal measured K+:Na+ ratios of 12--25:1. The cells were normally flat but addition of DBcAMP caused them to round up and form numerous processes, an appearance resembling that of astroglial cells in vivo. DBcAMP treatment also reduced the steady state levels of K+ measured with 86Rb+ by 15--30%, and had no effect on initial rates of 86Rb+ and 22Na+ uptake. The membrane potentials of cells treated with DBcAMP were studied, since only these were easily impaled. The membrane potentials of separate groups of cells gave means ranging from --65 to --75 mV at 35 degrees C, at an external K+ concentration ([K+]o) of 4.5 mM. The dependence of the membrane potentials of individual cells and groups of cells on [K+]o was studied. The slope of the potential per 10-fold change in [K+]o was 55--57 mV, at concentrations of K+ greater than 10--20 mM K+, and diverged from this slope at concentrations below this. This shows that these cells had some permeability to ions other than K+. Assuming that Na+ was the only other ion affecting the membrane potential, it was calculated that the permeability to Na+ was about 30 times less than K+. A similar result was obtained based on estimates of Na+ and K+ permeability from transport experiments on cells also treated with DBcAMP. The results obtained from these cells are compared to those found for other cultured glial cells and glial cells in vivo. We conclude that the membrane potentials of the cultured cells used in the present study show the closest resemblance so far to glia in vivo, since they are large and negative and are determined mainly by K+. However, the cultured cells have different properties from those reported in some studies for glial cells in vivo by showing free permeability to ions other than K+.

The effects of temperature and inhibitors on HCO3-stimulated swelling and ion uptake of monkey cerebral cortex.

In the presence of high concentrations of K+, additions of HCO3- as low as 0.35 mM caused a 23% increase in swelling, and concomitant increases in the chloride content of incubating monkey cerebrocortical slices. The uptake of chloride was accompanied by increased uptake of sodium and was highly temperature dependent, showing a marked activation at approximately 30 degrees C. A similar temperature activation was also found for a Mg2+-dependent, HCO3-stimulated ATPase activity in monkey cerebral cortex, consistent with a possible role for this enzyme in the K+ and HCO3-dependent swelling process and its associated ion movements. K+-dependent, HCO3-stimulated cerebrocortical tissue swelling with uptake of Na+ and Cl- was inhibited by acetazolamide indicating that carbonic anhydrase was also involved. The addition of ouabain also inhibited swelling and K+ and Cl- uptake at low concentrations, but led to increased swelling at higher concentrations ( greater than 10 mum). A similar biphasic effect on swelling was also seen following addition of ethacrynic acid.

Direct administration of methotrexate into the central nervous system of primates. Part 2: Distribution of 3H methotrexate after intrathecal lumbar injection.

The kinetics of distribution of 3H methotrexate (3HMTX) in the central nervous system, plasma, and urine after intraventricular, lumbar percutaneous puncture, and spinal catheter injections were compared. Levels of 3HMTX in whole brain after lumbar percutaneous injection were 40 times less than after intraventricular injection. Injection of 3HMTX via a spinal catheter increased the level of 3HMTX in whole brain but this was still tenfold less than after direct intraventricular instillation. Also, it was found that a disproportionately high amount of 3HMTX was in the brain-stem-cerebellum region which would further reduce the concentration of methotrexate in the cerebral hemispheres. Both intraventricular and lumbar spinal catheter administration of 3HMTX produced 3HMTX levels greater than 10(-6)M (moles/kg wet weight) in spinal cord tissue as measured by 3H specific activity between 2 to 8 hours after injection. Administration by lumbar percutaneous puncture, however, rarely resulted in this suggested therapeutic level of 10(-6)M. Initial 3HMTX levels in plasma after lumbar percutaneous instillation was 24 times greater than after intraventricular or lumbar spinal catheter injections. This indicated significant and unavoidable extradural leakage after lumbar percutaneous puncture, which may account for the substantially lower levels of 3HMTX in the brain and spinal cord tissue. It is concluded that intraventricular instillation of methotrexate is the best route of administering the drug to achieve therapeutic levels of methotrexate in both whole brain and throughout the spinal cord.

Evaluation of experimental spinal cord injury by measuring spontaneous spinal cord potentials.

The relationship between the spontaneous spinal electrogram and the degree of spinal cord injury was studied in anesthetic-free, surgically decerebrate cats that received experimental blunt trauma by the graded weight-drop method. It was found that the characteristic spontaneous slow negative potential of the spinal electrogram showed a frequency dependency that correlated positively with the intensity of the injury (impulse expressed in gm-sec). Graphs of the frequency of occurrence of the slow negative potentials as a function of time following initial injury indicated that both the slope and shape of the curve were dependent on the severity of the injury measured in gm-sec at the time of the injury and confirmed histologically. These results indicate that the spontaneous spinal electrogram may serve as a sensitive indicator of the degree of spinal cord injury and may be useful in the assessment of various treatment modalities.

Camurati-Engelmann disease (progressive hereditary craniodiaphyseal dysplasia). Case report.

In a patient with Camurati-Engelmann disease, orbital and optic nerve decompression resulted in improvement of papilledema. Subsequent x-ray films of the optic canals, however, revealed reconstitution of osseous optic canals bilaterally, and papilledema has returned in one eye. Definitive treatment of this dysplastic metabolic bone disorder rests in the control of rapid abnormal bone formation.

Analysis and measurement of some sources of variability in experimental spinal cord trauma.

Experiments were performed on anesthetized cats to determine whether the variability that is common in experimental spinal cord injuries produced by the weight-drop technique can be reduced if a more accurate determination of the actual intensity of the insult to the cord is measured. In addition, determinations of the contribution of such variables as mass of drop-weight, impounder diameter, and animal weight to variability were made. It was found that of the three measures of intensity readily available from a strain gauge transducer, impulse (change in momentum of drop-weight) showed the highest correlation with the histologically determined extent of the lesion. It was shown that the mass of the drop-weight had a significant effect on lesion size even when the gram X centimeter quantification of the injury was constant. Animal weight and impounder diameter did not make a significant contribution to the variability of injury as determined by low-power microscopy.

Direct administration of methotrexate into the central nervous system of primates. Part 1: Distribution and degradation of methotrexate in nervous and systemic tissue after intraventricular injection.

Levels of methotrexate (MTX) measured by both 3H radioactivity and dihydrofolate reductase assays were determined in cerebrospinal fluid (CSF), plasma, urine, and both neural and non-neural tissues at varying times after a single intraventricular injection into Cynomolgus monkeys (Macaca fascicularis). Clearance of the MTX from CSF was rapid after injection. A relatively constant level of 3HMTX was reached in plasma 2 1/2 hours after injection, and about 30% of the 3HMTX dose was excreted in the urine within 4 hours after injection. Maximum levels in CNS tissues were obtained by 4 hours after injection, and average concentrations of 10(-6) M MTX (moles/kg wet weight) were maintained in CSF for up to 12 hours and in brain for up to 24 hours after injection. Conversion of MTX to non-MTX products was detected in CSF between 4 and 12 hours, and in brain tissue between 12 and 24 hours after injection, and the amount of these products increased with time. Regional distribution studies in the cerebrum showed a U-shaped distribution curve for 3HMTX up to 12 hours after injection, which closely followed the 14C inulin distribution. Thus, the levels in deep cerebral tissue were less than the average level for brain, and this suggests that treatment of CNS tumors by intraventricular injection may have variable results, partly due to complex tissue distribution patterns.

Adenosine-stimulated astroglial swelling in cat cerebral cortex in vivo with total inhibition by a non-diuretic acylaryloxyacid derivative.

The intact cerebral cortices of cats were exposed in vivo under normothermic conditions and superfused with isotonic artificial cerebrospinal fluid containing added 0.125 mM adenosine. This resulted in chloridecation-rich cerebrocortical swelling which was shown by electron microscopy to be associated with an expanded astroglial compartment. The addition of DCPIB, a non-diuretic acylaryloxyacid analogue of ethacrynic acid and an inhibitor of coupled chloride-cation transport in cerebral cortex in vitro, totally blocked astroglial swelling and the concomitant increases in tissue ion contents. These studies support our previous experiments on the mechanism of formation of astroglial swelling. The pathological consequences of astroglial swelling and the clinical applications of these findings are discussed.

Delayed pulmonary dysfunction in head-injured patients.

Intracranial pressure (ICP), cardiopulmonary function, and the degree of neurological dysfunction were measured in 13 patients with serious head injury to determine the relationship of these indices to the development of delayed pulmonary dysfunction. All patients had serious isolated head injury with Glasgow Coma Scale scores of 7 or less 6 hours after injury and elevated ICP at the time of admission to the protocol. Three patients developed arterial pO2 of less than or equal to 80 torr despite the initiation of elevated inspired oxygen fraction (FIO2 greater than or equal to 0.5) and positive end expiratory pressure (greater than or equal to 5 cm H2O. One of these three patients had a decline in neurological function, quantified by the Albany Head-Injury Watch Sheet, associated with hypoxemia. The only patients who developed intrapulmonary shunt fractions of more than 15% were five patients who had increased pulmonary vascular resistance (PVR) and elevated or increasing cardiac index, suggesting persistent perfusion to areas of the lung which normally are hypoperfused due to hypoxic pulmonary vasoconstriction. This mismatching of the distribution of ventilation and perfusion was confirmed using the multiple inert gas elimination technique in two patients with an increased shunt fraction. Unperfused gas exchange units were also found to be present, as confirmed by an abnormal multiple inert gas elimination techniques, high PVR and dead space/tidal volume ratio (VD/VT), and low extravascular lung water. Abnormalities of ICP and cerebral perfusion pressure could not be correlated with changes in any of the cardiopulmonary functions studied.

Biology of glial swelling in experimental brain edema.

Most studies of clinically relevant cerebral edema emphasize the effect of added tissue fluid in white matter on gross distortion with transtentorial and subfalcine brain herniation. Our recent studies on altered tissue fluid compartmentation in cerebral gray matter suggest that significant microdistortion of relationships of capillaries to subserved tissue follows swelling of astroglia therein. Grave consequences to solute and gas exchange in focal regions may well be expected and are emphasized. The elucidation of the mechanisms of formation and inhibition of astroglial swelling by chemical agents, including chemically useful acylaryloxyacetic acid derivatives, are discussed. Furthermore, the effect of these agents in altering mortality and morbidity in a controlled, random study of animal head injury is presented.

Delayed impairment of arterial blood oxygenation in patients with severe head injury: preliminary report.

A drop in the arterial PO2 occurring 24 hours after head injury was identified in eight patients. Traditional modes of therapy include administration of supplemental oxygen and provision of an unobstructed airway. The latter proved to be inadequate to continually maintain the PaO2 at a level consistent with the O2 content of the inspired air. Initially, determination of the PaO2, after institution of supplemental oxygen, may demonstrate adequate oxygenation, but blood gas monitoring should be continued since a delayed fall in arterial oxygen tension may occur 24 hours after head injury. This period of potentially deficient blood oxygenation, if severe enough, may further aggravate preexisting brain damage and profoundly affect the ultimate outcome of the patient. The delayed fall in PaO2 is the result of intrapulmonary shunting principally due to a ventilation/perfusion mismatch. The precise mechanism of the ventilation/perfusion inequality in the brain-injured patient awaits further elucidation, but may differ from the alteration in pulmonary function seen in the Respiratory Distress Syndrome.