Characterization of pulmonary and myocardial beta-adrenoceptors with S-1'-[fluorine-18]fluorocarazolol

S-1'-[F-18]fluorocarazolol was administered to healthy volunteers to assess its potential for noninvasive measurement of regional pulmonary and myocardial beta-adrenoceptor densities. Methods: High-specific activity fluorocarazolol was intravenously injected on two separate occasions within a 1-wk interval. The initial injection was without pretreatment, but before the second injection, the volunteers either inhaled salbutamol (2 x 200 mu g aerosol) or they ingested pindolol (3 x 5 mg during a 12-hr interval). Twenty-eight PET time frames of 31 planes were acquired over a period of 60 min after each injection. Blood samples were drawn and analyzed for the presence of fluorocarazolol and radioactive metabolites. Results: Uptake of fluorocarazolol in the target tissues was hardly affected by salbutamol but was strongly depressed by pindolol. Pulmonary and myocardial tissue-to-plasma concentration ratios of fluorocarazolol reached plateau values of 11.6 +/- 0.6 (lungs) and 18.1 +/- 1.0 (heart) at 45-50 min postinjection. These values were reduced to 2.0 +/- 0.4 and 2.0 +/- 0.6 after treatment with pindolol. Conclusion: These data indicate that: 1. Pulmonary and myocardial uptake of radioactivity after intravenous administration of S-1'-[F-18]fluorocarazolol represents radioligand binding to beta-adrenoceptors. 2. Pulmonary binding occurs mainly in alveoli rather than in airway smooth muscle under these conditions. 3. Binding kinetics do not preclude quantification of receptors with compartment models

CAPILLARY ZONE ELECTROPHORESIS IONSPRAY MASS-SPECTROMETRY OF A SYNTHETIC DRUG PROTEIN CONJUGATE MIXTURE

Low-molecular-mass proteins, such as lysozyme, may be suitable carriers to target drugs to the kidney. Naproxen, an anti-inflammatory drug, has been conjugated with lysozyme via a covalent amide bond formed between the carboxylic acid function of naproxen and the amino group of one of the lysines in lysozyme. The reaction products were analysed by capillary electrophoresis-ionspray mass spectrometry. Native lysozyme and its conjugates with one, two and three naproxen molecules were separated and their identities were confirmed by mass spectrometry. The ion current profiles of the individual conjugates showed pH-dependent tailing and adsorption-desorption phenomena in the capillary electrophoresis column not observed in the total ion current profiles and not observable by UV detection

DRUG TARGETING TO THE KIDNEY WITH LOW-MOLECULAR-WEIGHT PROTEINS

This paper reviews the design of a drug targeting strategy for renal specific delivery and endorenal release of drugs using low-molecular-weight proteins (LMWPs). In general, LMWPs are known to be filtered and subsequently reabsorbed by the proximal tubular cells of the kidneys. Within these cells LMWPs are catabolized in lysosomes due to prevalence of proteolytic enzymes and pH of 4-5. As such, these LMWPs might serve as drug carriers that release drugs site-specifically in the kidneys. The ability of the kidney to release the parent drug from drug-LMWP conjugates and drug-spacer derivatives by enzymatic or chemical hydrolysis of the connecting bond has been tested by incubation experiments in renal cortex homogenates and lysosomal lysates. Drug-LMWP conjugates of naproxen, sulfamethoxazole and dopamine have also been studied in vivo. The pharmacokinetics of these conjugates have been examined in freely moving rats and compared to those obtained after injection of equivalent doses of unreacted mixtures of drug and protein. In addition, the disposition of the drug-conjugated LMWP has been studied by radioactive-imaging with a gamma-camera. These studies demonstrated that the kidneys are the main site of uptake of drug-conjugated LMWPs and that the drugs are renally uncoupled and finally excreted in the urine. gamma-Camera experiments revealed a predominant renal uptake of radio-iodinated drug-conjugated LMWPs. Coadministration of an excess of the native LMWP resulted in a pronounced decrease of urinary (parent) drug excretion, suggesting competition for proximal tubular uptake between the LMWP and the drug-conjugated LMWP. Endorenal parent drug release from the LMWP was demonstrated for drugs with carboxyl groups and amino groups. The NSAID, naproxen, was shown to be released from the LMWP conjugate in vivo, provided that coupling via its carboxyl group was performed via an ester bond using a L-lactic acid spacer. If naproxen was directly coupled to the protein via an amide linkage, renal proteolysis of the LMWP-conjugated drug gave rise to the formation of a naproxen-lysine derivative. Interestingly, this product has been demonstrated to be at least as active in inhibiting prostaglandin synthesis as naproxen itself. Sulfamethoxazole was shown to be endorenally released from a drug-LMWP conjugate, if its primary amino group was coupled to the LMWP via an acid-sensitive spacer. Apart from the altered kinetics of the drug itself, the selective renal distribution was further demonstrated by the absence of its hepatically formed metabolite N-4-acetylsulfamethoxazole in urine. This metabolite was abundantly present in the control experiments after injection of the drug in its uncoupled form. Parent drug regeneration in the kidney was further demonstrated for a dopamine-LMWP conjugate, linked through its amino group via a similar acid-sensitive spacer. Within a relatively low dose range of lysozyme, a dose-dependent endorenal dopamine release could be demonstrated. In principle, drugs released within the proximal tubular cell may leave this cell at the basolateral membrane (blood site) or at the brush-border membrane (urine site). Renal delivery of LMWP-conjugated naproxen resulted in detectable plasma levels of the parent drug. In contrast, in the case of LMWP-conjugated sulfamethoxazole and dopamine, no (increase in) plasma levels of the free drugs were measured

LOW-MOLECULAR-WEIGHT PROTEINS AS CARRIERS FOR RENAL DRUG TARGETING - NAPROXEN COUPLED TO LYSOZYME VIA THE SPACER L-LACTIC ACID

Low molecular weight proteins (LMWPs) are potential carriers for targeting drugs to the kidney. To test whether ester bonds are suitable for the reversible drug conjugation, the antiinflammatory drug naproxen (Nap) was conjugated to the LMWP lysozyme (LYSO) via an ester bond using an L-lactic acid spacer (Nap-lact-LYSO, 1:1:1). The distribution and degradation of the conjugate in rats were compared to those of an equimolar mixture of free drug and LMWP and of a directly coupled conjugate without spacer (Nap-LYSO). The plasma clearance of Nap-lact-LYSO closely resembled that of Nap-LYSO and LYSO itself. Its major accumulation site appeared to be the kidney as demonstrated by extracorporal gamma-camera counting of the LMWP. Renally released naproxen was excreted in the urine as 6-desmethyl-naproxen-sulfate (6-DMN-S). Apparently the kidneys represent the main sites of demethylation and sulfation after administration of the LMWP-coupled drug. In addition, the renal excretion of naproxen (including its metabolites) was significantly delayed and sustained as compared to that after injection of uncoupled naproxen. Using the L-lactic acid spacer LMWP conjugation, the renal selectivity of Nap was increased 5.6 +/- 0.41-fold. Additional in vitro studies with Nap-lact-LYSO revealed that renal generation of the parent drug coincided with formation of low molecular weight catabolites, mainly as naproxen-L-lactic acid-lysine (Nap-lact-Lys). This indicated that in vitro the rate of cleavage of the ester bond is significantly slower than digestion of the carrier backbone itself. It is concluded that for drugs with free carboxyl groups the coupling to LMWPs via alpha-hydroxy acids can result in renal-specific delivery and endorenal drug release

RENAL SPECIFIC DELIVERY OF SULFAMETHOXAZOLE IN THE RAT BY COUPLING TO THE LOW-MOLECULAR-WEIGHT PROTEIN LYSOZYME VIA AN ACID-SENSITIVE LINKER

Sulfamethoxazole (SM) was converted to a renal specific drug targeting preparation by coupling the drug to egg-white lysozyme via an acid-sensitive cis-aconityl linker (1:1). Due to this chemical manipulation SM was rapidly distributed to the kidney. Both in vitro and in vivo data indicate that SM was uncoupled from the carrier by chemical hydrolysis in the lysosomes of proximal tubular cells, resulting in parent active drug at the target site. This concept is applicable to other drug-polypeptide conjugates which rapidly distribute to the kidney and might enable selective manipulation of renal (patho)physiology