Synthesis of a series of stromelysin-selective thiadiazole urea matrix metalloproteinase inhibitors.

The synthesis and enzyme inhibition data for a series of thiadiazole urea matrix metalloproteinase (MMP) inhibitors are described. A broad screening effort was utilized to identify several thiadiazoles which were weak inhibitors of stromelysin. Optimization of the thiadiazole leads to include an alpha-amino acid side chain with variable terminal amide substituents provided a series of ureas which were moderately effective stromelysin inhibitors, with Ki's between 0.3 and 1.0 microM. The most effective analogues utilized an L-phenylalanine as the amino acid component. In particular, unsubstituted 46 had a Ki of 710 nM, while the p-fluoro analogue 52 displayed increased potency (100 nM). Stromelysin inhibition was further improved using a pentafluorophenylalanine substituent which resulted in 70, a 14 nM inhibitor. While gelatinase inhibition was generally poor, the use of 1-(2-pyridyl)piperazine as the amide component usually provided for enhanced activity, with 71 inhibiting gelatinase with a Ki of 770 nM. The combination of this heterocycle with a p-fluorophenylalanine substituent provided the only analogue, 69, with collagenase activity (13 microM). The SAR for analogues described within this series can be rationalized through consideration of the X-ray structure recently attained for70 complexed to stromelysin. Uniquely, this structure showed the inhibitor to be completely orientated on the left side of the enzyme cleft. These results suggest that thiadiazole urea heterocycles which incorporate a substituted phenylalanine can provide selective inhibitors of stromelysin. Careful selection of the amide substituent can also provide for analogues with modest gelatinase inhibition.

Synthetic matrix metalloproteinase inhibitors and tissue inhibitor of metalloproteinase (TIMP)-2, but not TIMP-1, inhibit shedding of tumor necrosis factor-alpha receptors in a human colon adenocarcinoma (Colo 205) cell line.

The solubilization of plasma membrane receptors through proteolytic cleavage of the ligand binding domain at the cell surface is an important mechanism for regulating cytokine function and receptor signaling. The inhibition of the shedding of a variety of receptors by synthetic inhibitors of the matrix metalloproteinases (MMPs) implicates metalloproteinases in this regulatory event. We examined the effects of two naturally occurring tissue inhibitors of metalloproteinases, TIMP-1 and TIMP-2, and several synthetic MMP inhibitors (MMPIs) on the shedding of both tumor necrosis factor alpha receptor type I (TNFalpha-RI; Mr 55,000) and TNFalpha-RII (Mr 75,000) by the Colo 205 human colon adenocarcinoma cell line. Culture of Colo 205 cells for 48 h resulted in the shedding of both TNFalpha-RI and TNFalpha-RII, as determined by ELISA. The shedding of TNFalpha receptors was not affected by TIMP-1 or protease inhibitors aprotinin, pepstatin, or leupeptin but was inhibited in a dose-dependent manner by the following synthetic MMPIs: batimastat and marimastat (BB-94 and BB-2516, respectively, British Biotech, Inc.); CT1418 (Celltech Therapeutics); CGS27023A (Novartis Pharmaceuticals); and RO31-9790 (Roche), with IC50s ranging from 3.2 to 38.0 microM. Similarly, TIMP-2 from two different sources reproducibly inhibited the shedding of both TNFalpha-RI and TNFalpha-RII in a dose-dependent manner (IC50 = 286 +/- 33 nM for TNFalpha-RI shedding and 462 +/- 52 nM for shedding of TNFalpha-RII). The inhibition of TNFalpha-RI shedding was confirmed in the SW626 human ovarian adenocarcinoma cell line. The synthetic MMPIs and TIMP-2, but not TIMP-1, also caused a dose-dependent increase in the number of TNFalpha receptors retained on the surface of Colo 205 cells, as determined by flow cytometry. Inhibition of TNFalpha receptor shedding with TIMP-2 occurs at molar concentrations 10-100 times less than those required with low molecular weight, synthetic MMPIs but at concentrations greater than those required to inhibit collagen degradation. Modulation of TNFalpha receptor shedding by TIMP-2 could have important implications for the pleiotropic effects of TNFalpha in both normal and malignant cells and for the pharmacological activity of synthetic MMPIs.

Use of gamma-glutamyl transpeptidase activity as a marker of hair cycle and anagen induction in mouse hair follicles.

gamma-glutamyl transpeptidase activity was monitored in cycling mice by histologic localization and biochemical assay. Our objective for this study is to establish the relationship between gamma-glutamyl transpeptidase activity and hair growth and to determine whether its activity can be correlated to induced hair growth. In cycling mouse skin, gamma-glutamyl transpeptidase activity is pronounced during anagen and greatly diminished during telogen. In the skin, the enzyme is present exclusively in the outer and inner root sheaths of hair follicles. gamma-glutamyl transpeptidase is limited to the follicle below the level of the sebaceous gland and is completely absent in the follicle above the sebaceous gland level. During anagen, the outer root sheath in the hypodermis is intensely positive for gamma-glutamyl transpeptidase activity whereas the hair matrix cells and dermal papillar are negative. The inner root sheath above the bulb shows distinctive membrane staining for gamma-glutamyl transpeptidase. gamma-glutamyl transpeptidase activity can be seen to vary only in cycling follicles. Inducing anagen by plucking hair shafts results in an increase in gamma-glutamyl transpeptidase activity directly correlated to hair regrowth. In a similar manner, mice were plucked and treated with a daily dose of 2% minoxidil. A slight difference in cycle lengths was seen in animals treated with minoxidil when compared to vehicle control. Minoxidil treatment may cause an early initiation of anagen, but both the minoxidil-treated skin and the vehicle-treated skin entered telogen at the same time. Together, these studies indicate that gamma-glutamyl transpeptidase is a specific marker of anagen in growing hair.

Efficacy of transnasal butorphanol tartrate in postepisiotomy pain: a model to assess analgesia.

Butorphanol tartrate, a synthetically derived opioid agonist-antagonist analgesic, was tested in a large group of postpartum women (N = 76) to assess the safety and analgesic efficacy of a recently approved transnasal preparation of this drug in the relief of postepisiotomy pain. The safety and efficacy of intravenous and intramuscular administration of butorphanol tartrate has been established over 14 years of clinical use. The new nasal spray dosage form offers a similar degree of efficacy with a rapid onset of action. Compared with the injectables and other drugs in this class, transnasal butorphanol has a longer duration of action (4 to 5 hours). In this double-blind, parallel-group, dose-response study, 76 female patients ages 17 to 37 years with moderate to severe postepisiotomy pain were randomly assigned to receive a single dose of transnasal butorphanol (0.25, 0.5, 1, or 2 mg) or placebo. The patients were evaluated for 6 hours. The results of the study indicate that the 1-mg and 2-mg doses were associated with greater efficacy compared with placebo using several markers for efficacy, including the pain relief score and time to remedication. The drug was well tolerated, dizziness and drowsiness being the most frequently reported adverse effects. Adverse effects appeared to be dose related.

A HPLC-based chloramphenicol acetyltransferase assay for assessing hair growth: comparison of the sensitivity of UV and fluorescence detection.

In our attempt to measure hair growth by hair-specific markers, we used transgenic mice to express the chloramphenicol acetyltransferase gene under the control of an ultrahigh sulphur keratin gene promoter. To quantitate expression of the keratin gene, we required a chloramphenicol acetyltransferase assay which could measure enzyme activity in a single follicle and also could be used to assay a large number of samples without loss of sensitivity. We achieved this objective by utilizing a fluorescent substrate for chloramphenicol acetyltransferase. With HPLC-fluorescence detection, this substrate provides a sensitivity of less than 1 x 10(-13) mol, which is 1000 times greater than that achievable with HPLC-UV detection in cultured follicles. Further, the assay was automated to facilitate the analysis of more than 100 samples/day. It should be possible to apply this fluorescent assay to a number of cell or tissue studies.

Potassium channel conductance: a mechanism affecting hair growth both in vitro and in vivo.

The opening of intracellular potassium channels has been suggested as a mechanism regulating hair growth. Enhancing the flux of potassium ions is a mechanism shared by several structurally diverse antihypertensive agents including minoxidil sulfate (the active metabolite of minoxidil), pinacidil, P-1075 (a potent pinacidil analog), RP-49,356, diazoxide, cromakalim, and nicorandil. Of these drugs, minoxidil, pinacidil, and diazoxide have been reported to elicit hypertrichosis in humans. This potassium channel hypothesis was examined by testing these drugs for effects on hair growth both in vitro and in vivo. For the in vitro studies, mouse vibrissae follicles were cultured for 3 d with drug and the effects on hair growth were measured by metabolic labeling. All drugs, except diazoxide, enhanced cysteine incorporation into the hair shafts of the cultured vibrissae. Diazoxide was poorly soluble and thus was tested only at low doses. Minoxidil, P-1075, cromakalim, and RP-49,356 were also evaluated in vivo by measuring hair growth effects in balding stumptail macaque monkeys. The drugs were administered topically to defined sites on balding scalps once per day for 4-5 months and the amount of hair grown was determined by monthly measurements of shaved hair weight. Three of the drugs produced significant increases in hair weight whereas, the RP-49,356 had no effect. These studies provide correlative evidence that the opening of potassium channels is an important regulatory mechanism for hair growth. This provides the impetus for further studies on this potentially important mechanism affecting hair biology.

Hair growth effects of oral administration of finasteride, a steroid 5 alpha-reductase inhibitor, alone and in combination with topical minoxidil in the balding stumptail macaque.

A 5 alpha-reductase inhibitor, finasteride, was administered orally at 0.5 mg/day, alone or in combination with topical 2% minoxidil, for 20 weeks to determine the effects on scalp hair growth in balding adult male stumptail macaque monkeys. A 7-day dose-finding study showed that both 0.5- and 2.0-mg doses of the drug produced a similar diminution in serum dihydrotestosterone (DHT) in male stumptails. Hair growth was evaluated by shaving and weighing scalp hair at baseline and at 4-week intervals during treatment to obtain cumulative delta hair weight (sum of the 4-week changes in hair weight from baseline) for the 20-week study. The activity of the 5 alpha-reductase enzyme was assessed by RIA of serum testosterone (T) and DHT at 4-week intervals. The combination of finasteride and minoxidil generated significant augmentation of hair weight (additive effect) compared to either drug alone. Finasteride increased hair weight in four of five monkeys. When the data of the one nonresponsive monkey were excluded, finasteride elicited a significant elevation in hair weight compared to topical vehicle alone. Minoxidil also evoked a significant increase in hair weight compared to vehicle alone. Serum T was unchanged, whereas serum DHT was significantly depressed in monkeys that received either finasteride or the combination of finasteride and minoxidil. These data suggest that inhibition of the conversion of T to DHT by this 5 alpha-reductase inhibitor reverses the balding process and enhances hair regrowth by topical minoxidil in the male balding stumptail macaque.

Liquid chromatographic determination of desfuroylceftiofur metabolite of ceftiofur as residue in cattle plasma.

A liquid chromatographic (LC) method has been developed for the determination of the desfuroylceftiofur metabolite of ceftiofur as a residue in the plasma of animals. Plasma sample in 0.1M pH 8.7 phosphate buffer containing dithioerythritol is incubated under nitrogen for 15 min at 50 degrees C. The sample is centrifuged, charged to a C18 cartridge, and washed with 0.1M ammonium acetate. The desfuroylceftiofur residue on the cartridge is derivatized by adding 0.1M ammonium acetate containing iodoacetamide and letting the cartridge stand in the dark for 30 min. The cartridge is then drained and rinsed, and the desfuroylceftiofur acetamide is eluted with methanol. The mixture is evaporated to dryness, dissolved in pH 10.6 sodium hydroxide, and charged to a SAX cartridge. The derivative is eluted with 2% acetic acid, reduced in volume, and dissolved in mobile phase for liquid chromatography. The LC system includes a C8 column and guard cartridge with UV detection at 254 nm. The gradient mobile phase (flow rate 1 mL/min) is 0.01M pH 5 ammonium acetate programmed to 29% methanol-water (60 + 40) in 25 min. Recoveries were 90-100% with a sensitivity of 0.1 ppm or less. The procedure has been applied to the plasma of cattle, rats, horses, pigs, and dogs.

Clearance of triamcinolone from vitreous.

The clearance of intravitreally injected triamcinolone acetonide was monitored by both indirect ophthalmoscopy and high-performance liquid chromatography (HPLC) and found to be more rapid than previously reported by others. Twenty-four rabbits were intravitreally injected in each eye with 0.4 mg of triamcinolone acetonide. Three rabbits each were sacrificed at intervals ranging from one hour to 46 days. The vitreous was then harvested and processed for HPLC analysis. Triamcinolone and the internal standard prednisolone were identified and quantitated by the use of HPLC, which was found to be both sensitive and specific for the steroids. The half-life as determined by HPLC was 1.6 days, the level at 13 days postinjection was 66 +/- 19 micrograms, and no drug was detectable by HPLC analysis at 21 days in five of six eyes. The intravitreal masses thought to be triamcinolone were clinically observable to an average of 23.3 days. The chromatographically determined clearance rate did not correlate well with clinical impressions based on indirect ophthalmoscopy.

Liquid-chromatographic quantification of piracetam.

Piracetam, an analog of gamma-aminobutyric acid, absorbs maximally at 197 nm. Its molar absorptivity at 208 nm and pH 4.5 is 3576 (SD 251) L . mol-1 cm-1, approximately 45% of its absorptivity at 197 nm. Direct quantification of piracetam at 197 nm in biological extracts is complicated by the fact that many other compounds absorb between 190 and 220 nm due to carbon-nitrogen bonding. Chromatography of methanol extracts of serum and aqueous humor on a reversed-phase C-18 column developed isocratically with KH2PO4 (0.1 mol/L, pH 4.8) allows detection and quantification of 0.2 mmol of piracetam per liter. Under these conditions the retention time of piracetam is about 5 min. The detector response is linear for quantities between 5 and 15 nmol. The method is rapid, inexpensive, and convenient for the clinical laboratory.