Alfred André Hofmann

Identification of (−)-Δ9-6a,10a-trans-tetrahydro-cannabinol and two of its metabolites in rats by use of combination gas chromatography-mass spectrometry and mass fragmentography

Abstract Two metabolites of (−)- Δ 9 -6 a ,10 a - trans tetrahydrocannabinol (Δ 9 THC), one of the psychotomimetic components of Cannabis sativa L, were found after this substance was injected intraperitoneally into rats. Extracts of rat urine, blood, bile and feces were analysed by gas chromatography, mass spectrometry and mass fragmentography. In urine and feces unchanged Δ 9 THC was found. After glucuronidase treatment of feces, cannabinol (CBN) and an equal amount of Δ 9 THC was also found. Dihydroxy-Δ 9 -THC was identified in urine and feces. Diacetyl-Δ 9 THC was isolated from bile.

«Affinity Labelling» des cholinergischen Rezeptors der motorischen Endplatte mit Diazo-Acetylcholin

Diazoacetylcholinebromide was used for affinity labelling of the cholinergic receptors of endplates in the mouse diaphragm. Under irradiation by light (Hg-high-pressure-lamp), irreversible depolarizing block of neuromuscular transmission developed. Acetylcholinesterase was influenced only by much higher concentrations, which demonstrates different localizations of the active enzyme center and the cholinergic receptor.

KATP CHANNEL OPENERS REVERSE IMMUNE COMPLEX-INDUCED AIRWAYS HYPERREACTIVITY INDEPENDENTLY OF SMOOTH MUSCLE RELAXATION

Many openers of ATP-dependent potassium channels (KATP channel openers) cause bronchorelaxation, whereas only a few of them have been claimed to reverse airways hyperreactivity. We investigated whether the antihyperreactive effect is a general feature of KATP channel openers and whether this property is linked to their ability to relax airways smooth muscle. For this purpose, the potency of the four KATP channel openers, bimakalim, rilmakalim, levcromakalim and SDZ PCO 400 ((−)-(3S,4R)3,4-dihydro-3-hydroxy-2,2-dimethyl-4-(3-oxo-cyclopent-lenyloxy)-2H-1-benzopyran-6-carbonitrile), to inhibit bombesin-or histamine-induced bronchoconstriction and to reverse immune complex-induced airways hyperreactivity to histamine in guinea pigs, was compared to salbutamol, following intratracheal administration to minimize pharmacokinetic differences.Total lung resistance (RL) was determined in anaesthetized, ventilated guinea pigs. Bronchoconstriction, measured as increase in RL, was elicited in normoreactive animals by i.v. infusion of bombesin (100 ng/kg/min) or by i.v. injection of histamine (1.8–10 µg/kg). Airways hyperreactivity was induced by acute i.v. administration of preformed immune complexes. I.v. bolus injections of histamine were used to define the sensitivity of the airways prior to and after the exposure to immune complex.Levcromakalim (ED50 = 150 μg/kg), bimakalim (ED50 = 4 μg/kg), rilmakalim (ED50 = 40 μg/kg) and SDZ PCO 400 (ED50 = 280 μg/kg) reversed bombesin-induced bronchoconstriction with lower potency than salbutamol (ED50 = 1 μg/kg). The four KATP channel openers and salbutamol also reversed immune complex-induced airways hyperreactivity to histamine with ED50 values which were markedly lower than those for reversal of bombesin-induced bronchoconstriction; the rank order of potency was rilmakalim (ED50 = 0.2 μg/kg) > bimakalim (ED50 = 0.5 μg/kg) > SDZ PCO 400 (ED50 = 3.2 μg/kg) > levcromakalim (ED50 = 22 μg/kg). Salbutamol (ED50 = 0.008 μg/kg) was the most potent compound in this test. Bimakalim, levcromakalim and SDZ PCO 400 did not inhibit histamine-induced bronchoconstriction in normoreactive guinea pigs at doses which completely reversed immune complex-induced airways hyperreactivity to histamine. For rilmakalim and salbutamol, 60–130 times higher doses were needed for protection against histamine-induced bronchoconstriction in normoreactive guinea pigs than for reversal of airways hyperreactivity. There was a poor correlation between the ED50 values for inhibition of histamine- or bombesin-induced bronchoconstriction in normoreactive guinea pigs and the reversal of immune cimplex-induced airways hyperreactivity. It is thus concluded that the ability of KATP channel openers to reverse immune complex-induced airways hyperreactivity is independent of their ability to reverse or prevent bronchoconstriction and thus from their ability relax airway smooth muscle.

IN VITRO AND IN VIVO EFFECTS OF SCA40 ON GUINEA PIG AIRWAYS

Abstract SCA40 (6-bromo-8-methylaminoimidazo[1,2-a]- pyrazine-2-carbonitrile), a compound which had been described as an opener of Ca2+-dependent large conductance potassium channels (BKCa channels), was investigated in comparison with salbutamol for in vitro and in vivo bronchospasmolytic effects and for the ability to reverse airways hyperreactivity in guinea pigs. SCA40 reduced the spontaneous tone of isolated guinea pig tracheal rings with a biphasic concentration-response curve (first phase: pD2 = 8.0, EMax = 29.7% of maximal effect; second phase: pD2 = 6.4, EMax = 72.6%). The salbutamol curve was monophasic (pD2 = 8.0, EMax = 100%).Total lung resistance (RL) was determined in anaesthetized, ventilated guinea pigs. Bronchoconstriction, measured as an increase in RL, was elicited in normoreactive animals by i.v. infusion of bombesin (100 ng/kg/min) or by i.v. injection of histamine (1.8–5.6 μg/kg). Airways hyperreactivity was induced by acute i.v. administration of pre-formed immune complexes. Intravenous bolus injections of histamine (2.4 μg/kg) were used to define the sensitivity of the airways prior to and after the exposure to immune complex. Following intratracheal (i.t.) administration, SCA40 reversed bombesin-induced bronchoconstriction with an ED50 of 43 μg/kg (EMax = 57%). The ED50 for salbutamol was 0.8 μg/kg i.t. (EMax = 78%). Histamine-induced bronchoconstriction in hyperreactive guinea pigs was inhibited by SCA40 with an ED50 of 13 μg/kg i.t. (EMax = 82%). Salbutamol completely inhibited histamine-induced bronchospasm with an ED50 of 9 ng/kg i.t. In normoreactive guinea pigs, SCA40 prevented histamine-induced bronchoconstriction with an ED50 of 100 μg/kg i.t.; for salbutamol the ED50 in this test was 0.48 μg/kg i.t. Thus, for both SCA40 and salbutamol, the effects obtained at low doses in hyperreactive guinea pigs represent a true reversal of airways hyperreactivity, whereas at higher doses, anti-hyperreactive and bronchospasmolytic properties may account for the observed effects.In conclusion, SCA40 relaxes guinea pig airways smooth muscle in vitro and in vivo, and it partly reverses airways hyperreactivity. With respect to both potency and efficacy, SCA40 is markedly less active than the β-adrenoceptor agonist salbutamol.

Synthesis of specific cholinergic inhibitors for affinity chromatography

Our first aim was to simplify the spacer-synthesis for affinity-chromatography of cholinergic proteins. Further-more we synthesized 2 new inhibitors which proved to be useful for purification of acetylcholine-receptor protein.

Cholinergic Receptor Isolation

The isolation of specific cholinergic bindin protein (acetylcholine receptor = AChR) has been achieved successfully by numerous research groups (1-4, 7-9, 11, 12). The ideal source for such preparations was the electric organ of several species of Torpedo. All successful preparations notwithstanding, there are still some serious problems waiting to be solved. The first problem is the stability of AChR. Compared to acetylcholinesterase (AChE) which can be kept at room temperature for several days without losing enzymatic activity, the AChR is less stable. Regarding stability as shown by immunological properties only, the AChR is remarkably stable. Even methods such as freeze drying of the electric organ or protein fractions during preparation, binding to animal toxins (mostly snake venoms) and application of drastic methods to release it from affinity columns do not interfere with the immunological properties. Regarding stability in terms of pharmacological properties, however, the AChR is very unstable and continuously loses its binding properties during isolation and over the course of time. It is still an open question whether a free isolated receptor in solution will ever behave like a receptor in its natural environment embedded in membranes. Some factors which might interact with this very sensitive protein are 1) ambient oxygen, 2) proteolytic enzymes (a number of proteolytic enzymes are liberated in the course of isolation), 3) microbes, 4) acetylcholine (ACh) (reasonable amounts of ACh are liberated during preparation), and 5) other agonist type molecules (small molecules with depolarizing ability have been used for affinity chromatography and/or elution from the affinity column.

Affinity-labelling of the muscarinic receptor of the guinea-pig ileum

Summary1.The activities of some cholinergic compounds were investigated on the terminal ileum of the guinea-pig and their muscarinic potencies were found to be in the following order: acetylcholine (ACh) > diazoacetylcholine (DACh) > iodoacetylcholine (IACh) > azidoacetylcholine (AACh).2.Protection experiments with atropine showed a decreased affinity of the four cholinergic compounds for the muscarinic receptor, demonstrating a direct interaction of the drugs and the receptor.3.The maximal contractions (intrinsic activity) caused by DACh were greater than those caused by ACh, IACh and AACh.4.Incubation of the ileum with ACh was followed by a reversible loss of its ability to contract (desensitization). The time course of recovery was similiar to that after incubation with AACh.5.IACh caused a partial (50%), irreversible paralysis of the muscle.6.The furaziridines seem to react partially irreversibly (30%), whereas, paranitrophenyldiazonium fluoborate (p-NDB) caused a complete, irreversible blockade of the muscarinic receptor.