Bone affinity of a bisphosphonate-conjugated protein in vivo.

Growth factors capable of stimulating bone formation are potential therapeutic agents for osteoporosis treatment. It is essential, however, that a targeting mechanism is incorporated into the growth factors to deposit them at osseous tissue with minimal distribution to extraskeletal sites. To this end, a strategy has been developed in which a bone-seeking molecule, 1-amino-1,1-diphosphonate methane (aminoBP), was chemically conjugated to a model protein, bovine serum albumin (BSA). This study was carried out to assess the bone affinity of the conjugates in a tibia injection model. Using ovariectomized (OVX) rats, initial (3 h) retention of BSA and aminoBP-BSA were found to be equivalent when injected into the medullary cavity of tibia. After 1 day, an 8- and 12-fold higher tibiae retention of the protein was obtained in normal and OVX rats as a result of aminoBP conjugation. A similar result ( approximately 12-fold difference) was also obtained in OVX rats after 3 days. We concluded that aminoBP conjugation to BSA imparted a high bone affinity and enhanced bone retention of proteins in normal and OVX rats.

Bisphosphonate conjugation to proteins as a means to impart bone affinity.

Growth factors are endogenous proteins capable of stimulating new bone formation, but their clinical benefit for systemic stimulation of bone mass has not been demonstrated. The critical challenge is to deliver a significant dose of the proteins to bone after intravenous injection. This challenge may be overcome by derivatizing proteins with ligands that exhibit a high bone affinity (e.g., bisphosphonates). To demonstrate the feasibility of this approach, 1-amino-1,1-diphosphonate methane (aminoBP) was conjugated to a model protein, albumin. The conjugation was performed by (1) converting the amino group of aminoBP to a thiol group using 2-iminothiolane, (2) derivatizing the albumin amino groups with a thiol-reactive sulfosuccinimidyl-4-(N-maleimidomethyl)-1-cyclohexane carboxylate, and (3) reacting the derivatized albumin with thiolated aminoBP. Typically, 1-4 aminoBP molecules per albumin were obtained. The conjugated albumin exhibited a high affinity to hydroxyapatite that was proportional to the extent of conjugation. The conjugates were shown to exhibit a high affinity to bone matrix in vitro in a serum-containing medium. Once bound to bone matrix, the conjugates were found to desorb more slowly than the unmodified albumin, especially from bone whose organic matrix was removed by ashing. In conclusion, conjugation of bisphosphonates to albumin was shown to impart a high bone affinity to the protein, and such conjugates can be potentially targeted to bone.

Treatment and survival study in the C57BL/6J-APC(Min)/+(Min) mouse with R-flurbiprofen.

Our previous studies with the mouse model of familial adenomatous polyposis (FAP), C57BL/6J-APC(Min)/+ or Min mouse, demonstrated the optimal dose for adenoma reduction with R-flurbiprofen was 10 mg/kg/day as an undivided dose. Divided doses exhibited no increased efficaciousness. This study examines 10 mg/kg R-flurbiprofen daily (qd) on survival as well as a second daily (q.o.d.) schedule and compares it with sulindac sulfone. The q.o.d. schedule at 10 mg/kg was equally efficacious as qd treatment at the same dose. For the q.o.d. group, tumor number decreased similarly (p<0.01); while body weight gain (p<0.01), hematocrit and average tumor area (both, p<0.05) were improved compared with qd treatment. Treatment with R-flurbiprofen (10 mg/kg/day) increased survival significantly (p=0.0004, log-rank) compared to vehicle treated animals. Major biological endpoints (hematocrit, weight gain, tumor number, average and total area [99% reduction]) were significantly improved in treated animals (p<0.01). Sulindac sulfone treatment (50 mg/kg/day) of the Min mouse produced no significant biological benefit. The dose schedule study suggests that for tumor reduction it is necessary to attain a threshold drug-level but not necessarily sustain it over 24 hrs (pharmacodynamic t1/2 >> pharmacokinetic t1/2). During the period of administration R-flurbiprofen dramatically prolongs survival for the mouse model of the human disease, FAP.

The algorithm for cross-evaluation (ACE) of biologically active compounds.

A non-linear algorithm, ACE (Algorithm for Cross-Evaluation), was developed to correlate the activity of anti-cancer drugs against human cell lines. ACE evaluates the relationships among compounds and cell lines and displays the resulting clusters on a 3-D surface plot. This plot along with a frequency plot and rank graph provides clear information regarding the sensitivity of groups of cell lines to particular compounds. The program was tested on published anti-cancer data sets and found associations useful in the selection and design of chemotherapy drugs for pre-clinical trials. ACE may be applied to other situations where details of groupings and relationships need to be extracted from large sets of data.

Mechanism of enhancement of intestinal ulcerogenicity of S-aryl propionic acids by their R-enantiomers in the rat.

We previously observed a marked increase in gastrointestinal toxicity of rac-flurbiprofen compared to the therapeutically equivalent dose of the S enantiomer. This paper quantitates these observations and examines the mechanism by which this paradoxical toxicity occurs. We have evaluated the ulcer scores, mucosal neutrophil infiltration, by immunostaining of CD11/18 antigen, and mucosal neutrophil activity by myeloperoxidase measurement at two dose levels of (R)-, (S)-, and rac-flurbiprofen, administered over 30 days. Dose-response for intestinal ulcer production was observed for rac- and (S)-flurbiprofen; animals given (R)-flurbiprofen exhibited no ulcers. Yet rac-flurbiprofen proved to be twice as ulcerogenic as (S)-flurbiprofen. The mechanism of the exacerbation of gastrointestinal toxicity of (S)-flurbiprofen by the noncyclooxygenase inhibiting (R)-flurbiprofen is believed to be associated with its effect on ICAM-1 up-regulation. This is followed by neutrophil adhesiveness to ICAM-1 via the LFA-1 antigen on its surface and the extravasation of neutrophils into the tissue. We also examined the effect of high dose (R)-flurbiprofen vs vehicle over 15 days in animals in which ulcers had been produced by treatment with (S)-flurbiprofen for the previous 15 days. (R)-flurbiprofen did not sustain induced ulcers. The results of this study suggest that human studies be conducted to determine if enhanced gastrointestinal toxicity occurs in man. This is at issue since rac compounds of this class are available over the counter and others may be introduced.

R-flurbiprofen chemoprevention and treatment of intestinal adenomas in the APC(Min)/+ mouse model: implications for prophylaxis and treatment of colon cancer.

We used the C57BL/6J-APC(Min)/+ mouse (Min mouse) to evaluate the chemopreventive effects of R-flurbiprofen (R-FB), the noncyclooxygenase-inhibiting enantiomer of FB. Weanling Min mice were administered 6 weeks of oral treatment with R-FB using 2.5-25 mg/kg of R-FB once per day (q.d.), 2.5-10 mg/kg of R-FB twice per day (b.i.d.), and 5 mg/kg of R-FB b.i.d. challenged with a high saturated fat diet. At necropsy we determined tumor and ulcer numbers, tumor size, and plasma levels of R- and S-FB. A linear dose response was observed from 2.5 to 10 mg/kg of R-FB, regardless of whether the drug was administered as a single or divided dose. Reductions in tumor number were significant (P < or = 0.02) for doses of R-FB from 2.5 to 25 mg/kg/day. A dose of 5 mg/kg R-FB b.i.d. was able to overcome the doubling in tumor number associated with the high saturated fat diet. At 20 and 25 mg/kg/day R-FB, we obtained the maximum response with up to 90% inhibition of total tumor number. At these doses, however, there was toxicity and animal deaths. This toxicity was associated with ulceration, presumably resulting from the in vivo epimerization of R- to S-FB that occurs in the mouse. Thus, we evaluated the oral pharmacokinetics of R-FB and its conversion to S-FB in wild-type mice. These kinetics experiments revealed inversion rates of 7.3 and 11.0% for the 2.5 and 10 mg/kg R-FB doses, respectively. S-FB administered alone (0.5 and 2.0 mg/kg q.d.), in doses mimicking the concentrations of S-FB associated with the R to S epimerization of the doses of R-FB used in our experiments, had little or no antitumor efficacy (P > 0.05). Thus, we conclude that R-FB itself, not the S-FB resulting from epimerization in the mouse, inhibits adenoma formation in the Min mouse. In humans, where there is no R to S epimerization, it is possible that larger doses of R-FB can be used without causing cyclooxygenase inhibition and its resulting ulcerogenicity and other side effects. To assess the effect of R-FB on established adenomas, we allowed 40 Min mice to remain untreated until 70 days of age (the time of necropsy in the previous experiments) and then treated them for an additional 42 days with 10 mg/kg R-FB q.d. or 5 mg/kg R-FB b.i.d.. Both drug-treated groups demonstrated tumor numbers significantly less than that of the vehicle control (P < 0.01). Our results suggest that prophylaxis and treatment trials of R-FB should be extended to humans.

Endogenous natriuretic factors 6: the stereochemistry of a natriuretic gamma-tocopherol metabolite LLU-alpha.

2,7,8-Trimethyl-(S)-2-(beta-carboxyethyl)-6-hydroxy chroman (S-LLU-alpha) isolated from human uremic urine is apparently an oxidative side-chain degradation product of gamma-tocopherol. This compound exhibits natriuretic activity in vivo and it appears to mediate the inhibition of the 70 pS K+ channel in the apical membrane of the thick ascending limb of the nephron. The stereochemistry at the C-2 of LLU-alpha has been unequivocally established to be S(+) by X-ray crystallographic analysis of a diastereomeric amide derivative. It was also established that the chroman ring oxidation of S-LLU-alpha proceeded without racemization at C-2. This finding can be extended to nonepimerization at C-2 of alpha-delta tocopherols (Vitamin E) during side-chain oxidation and stereospecificity (retention or inversion) of oxidative opening of the chroman ring. The resolution of the enantiomers of the parent compound and derivatives was accomplished by chiral high-performance liquid chromatography. The stereospecific enzymatic hydrolysis by an array of commercially available enzymes of the racemic methyl ester of LLU-alpha was investigated. The lipase from Humicola languinosa appears to be the best enzyme for resolution by selective hydrolysis of the racemic methyl ester.

Endogenous natriuretic factors 7: biospecificity of a natriuretic gamma-tocopherol metabolite LLU-alpha.

The structural elucidation and mechanism of action of a potential component, LLU-alpha, of what is possibly a multifactorial complex known as "natriuretic hormone" was recently reported [Wechter, W.J. et al. (1996a) Proc. Natl. Acad. Sci. U.S.A. 93: 6002-6007]. "Natriuretic hormone," a long-sought factor, is believed to regulate extracellular fluid volume and consequently be pathomimetic for hypertension, cirrhosis, congestive heart failure and other volume expanded states. The studies reported herein further characterize LLU-alpha. The precursor of the endogenous LLU-alpha was demonstrated to be gamma-tocopherol by radiolabeling studies. The pharmacokinetics of infused rac-LLU-alpha proved to be biphasic (half-lives: 12 min and 6 h). Specificity of the inhibition of the 70 pS potassium channel of the thick ascending limb of the loop of Henle was examined with the natural S-enantiomer being the most potent known inhibitor whereas the analogous alpha-tocopherol metabolite, rac-5-Me-LLU-alpha, showed no inhibition. Rac-LLU-alpha does not inhibit two isozymes of the Na+/K+-ATPase. LLU-alpha is natriuretic acting via inhibition of the 70 pS potassium channel and not Na+/K+-ATPase, the assumed mechanism of action of the "natriuretic hormone." LLU-alpha, a metabolite of a vitamin, if it were found to play a role in the regulation of extracellular fluid volume, would be the second example of a vitamin acting as a precursor for a hormone. Of considerable interest is the fact that this manuscript reports the first biological activity of gamma-tocopherol, a member of the vitamin E complex.

Calculation of inversion half-lives of aryl propionic acid class nonsteroidal antiinflammatory drugs.

Although inversion of the R-enantiomers of the aryl propionic acid (APA) class of nonsteroidal antiinflammatory drugs (NSAIDs) in humans and other mammals has been known for more than 20 years, no satisfactory method has been developed for evaluating the half-life of the inversion process. This parameter is useful in assessing the pharmacodynamic contribution of the R-prodrug to the cyclooxygenase-inhibiting S-enantiomers. Further, it provides a similar means of evaluating the analgesic contributions of R-enantiomers to racemic mixtures. Using human and animal data, we have mathematically determined the inversion half-life (t1/2i) for ibuprofen, ketoprofen, and fenoprofen. The relation of the sensitivity of this value to the terminal half-life (t1/2) of the R-enantiomers of APA class drugs also is discussed.

Antiproliferative effects of the enantiomers of flurbiprofen.

Nonsteroidal antiinflammatory drugs (NSAIDs) are recognized for inhibiting growth of colon tumors in animal models, and for reducing the risk of colon cancer in humans. The mechanisms involved have not been established, but are thought to be related to reduced prostaglandin biosynthesis. The present study investigates the effect of COX-inhibiting and non-COX-inhibiting enantiomers of flurbiprofen on rat colonocyte proliferation. Intestinal ulceration was used as a surrogate indicator of COX inhibition. Sprague Dawley rats were treated orally with 6.3 mg/kg of R- or s-flurbiprofen or vehicle. Colonocyte labeling index and small bowel ulcer index were measured. R-flurbiprofen and S-flurbiprofen significantly reduced colonocyte labeling index, by 34% and 23% respectively, compared with vehicle. R-flurbiprofen caused minimal ulcer formation (4.48 mm2) compared with S-flurbiprofen (94.4 mm2). These findings suggest that R-flurbiprofen-mediated control of colonocyte proliferation is independent of prostaglandin biosynthesis.

A new endogenous natriuretic factor: LLU-alpha.

For over three decades, renal physiology has sought a putative natriuretic hormone (third factor) that might control the body's pool of extracellular fluid, an important determinant in hypertension, congestive heart failure, and cirrhosis. In our search for this hormone, we have isolated several pure natriuretic factors from human uremic urine that would appear, alone or in combination, to mark a cluster of phenomena previously presumed to be that of a single "natriuretic hormone." This paper reports the purification, chemical structure, and total synthesis of the first of these compounds, LLU-alpha, which proved to be 2,7,8-trimethyl-2-(beta-carboxyethyl)-6-hydroxychroman, presumably a metabolite of gamma-tocopherol. Both natural LLU-alpha and synthetic material are identical (except for optical activity) with respect to structure and biological activity. It appears that the natriuretic activity of LLU-alpha is mediated by inhibition of the 70 pS K+ channel in the apical membrane of the thick ascending limb of the kidney.

Endogenous natriuretic factors. 5. Synthesis and biological activity of a natriuretic metabolite of diltiazem and its derivatives.

In our search for endogenous natriuretic factors from human uremic urine, we have previously identified a new metabolite of the drug diltiazem (Murray et al. Life Sci. 1995, 57, 2145-2161). The structure of this metabolite, (+)-(2S,3S)-3-hydroxy-5-(2-hydroxyethyl)-2,3-dihydro-2-(4-methoxyphenyl) -1,5-benzothiazepin-4(5H)-one (LLU-beta1; 2), was proved by unequivocal synthesis from a diltiazem synthon. The synthetic material also proved to be natriuretic as had the urinary isolate. Given the acetylation at C-3 in diltiazem, the 3-monoacetate (8) and diacetate (3) derivatives of 2 were prepared. The 4-nor-keto (6) derivative of 2 was also synthesized. Only the parent 2 induced natriuresis over a range of doses without accompanying kaliuretic activity at some doses.

Synthesis and bioactivity studies of 1-adamantanamine derivatives of peptides.

Small enkephalin-related peptides containing a 1-adamantanamine moiety coupled through an amide linkage at the C-terminus were synthesized. Several of the compounds showed high mu opioid activity and mu receptor selectivity. The new adamantanamine derivatives were also examined for antiviral activity against HIV-1 in a cell culture system. Some of them inhibited syncytia formation even when the antigen assay gave evidence for viral replication.

Endogenous natriuretic factors 3: isolation and characterization of human natriuretic factors LLU-alpha, LLU-beta 1, and LLU-gamma.

A low molecular weight endogenous substance believed to be responsible for extracellular fluid homeostasis in mammals has been sought for many years. Our goal is to isolate and structurally characterize this putative "natriuretic hormone." We have developed an assay using the conscious rat to measure prolonged natriuresis (Benaksas et al (1993) Life Sciences, 52, 1045-1054), the activity originally described for this putative substance. Using this assay we have identified a number of natriuretic compounds isolated from human uremic urine. The collected urine is processed by ultrafiltration (< or = 3 kDa), gel filtration chromatography (G-25) and extraction with isopropanol and diethyl ether. The organic soluble material is then subjected to sequential high-performance liquid chromatography. We report here the initial characterization of two pure isolates (LLU-alpha and LLU-gamma) obtained by this method, and the structural elucidation of a third pure compound, LLU-beta 1, a natriuretic and previously unreported metabolite of the drug diltiazem.

Isolation of the nickel-chlorin chelate tunichlorin from the South Pacific ocean sea hare Dolabella auricularia.

The Papua New Guinea shell-less mollusc Dolabella auricularia has been found to contain a series of green to blue-green chlorins. One of these compounds was found to be the nickel chelate tunichlorin [1] which was isolated previously only from the Caribbean tunicate Trididemnum solidum. Discovery of tunichlorin [1] in a sea hare suggests that its occurrence in algae-consuming marine animals may be more common than earlier realized, and it may have a role in electron transfer or other metabolic processes.

Complexes of Fe(III) with amino sugars and small glycopeptides.

The complexes of Fe(III) with small molecules (glycopeptides, amino sugars) related to peptidoglycan monomer (PGM) structure were prepared and characterized by chemical and physicochemical methods. A simple method for preparation using separation of the reaction products on a molecular sieve was introduced. Using this method, monomeric complexes of small molecular weight were obtained. The likely structures were proposed on the basis of analytical results.

A convenient synthetic route to the disaccharide repeating-unit of peptidoglycan.

Glycosylation of the readily accessible benzyl 2-acetamido-6-O-benzyl-2-deoxy-3-O-[(R)-1-(methoxycarbonyl)ethyl]-alpha- D- glucopyranoside with 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-beta-D-glucopyranosyl chloride (2), using the silver triflate method in the absence of a base, afforded 65-70% of the fully protected [beta-D-GlcNPhth-(1----4)-MurNAc] methyl ester derivative 4, the structure of which was ascertained on the basis of 500-MHz 1H-n.m.r. data. 2,2'-Dideoxy-2,2'-diphthalimido-beta,beta-trehalose hexa-acetate was a by-product. Removal of the Phth group from 4, followed by acetylation, yielded 90% of the acetylated 1,6-di-O-benzyl derivative 5, which, on saponification and catalytic hydrogenation, afforded 2-acetamido-4-O-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-3-O-[(R)-1- carboxyethyl]-2-deoxy-D-glucopyranose. Similarly, 5 was converted into the acetylated methyl ester derivative, which, on selective removal of the methyl ester group, gave benzyl 2-acetamido-4-O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-beta-D- glucopyranosyl)-6-O-benzyl-3-O-[(R)-1-carboxyethyl]-2-deoxy-alpha-D- glucopyranoside. An alternative route for the preparation of 2 is described.