Multiparametric MRI of solid renal masses: pearls and pitfalls.

Functional imaging [diffusion-weighted imaging (DWI) and dynamic contrast enhancement (DCE)] techniques combined with T2-weighted (T2W) and chemical-shift imaging (CSI), with or without urography, constitutes a comprehensive multiparametric (MP) MRI protocol of the kidneys. MP-MRI of the kidneys can be performed in a time-efficient manner. Breath-hold sequences and parallel imaging should be used to reduce examination time and improve image quality. Increased T2 signal intensity (SI) in a solid renal nodule is specific for renal cell carcinoma (RCC); whereas, low T2 SI can be seen in RCC, angiomyolipoma (AML), and haemorrhagic cysts. Low b-value DWI can replace conventional fat-suppressed T2W. DWI can be performed free-breathing (FB) with two b-values to reduce acquisition time without compromising imaging quality. RCC demonstrates restricted diffusion; however, restricted diffusion is commonly seen in AML and in chronic haemorrhage. CSI must be performed using the correct echo combination at 3 T or T2* effects can mimic intra-lesional fat. Two-dimensional (2D)-CSI has better image quality compared to three-dimensional (3D)-CSI, but volume averaging in small lesions can simulate intra-lesional fat using 2D techniques. SI decrease on CSI is present in both AML and clear cell RCC. Verification of internal enhancement with MRI can be challenging and is improved with image subtraction. Subtraction imaging is prone to errors related to spatial misregistration, which is ameliorated with expiratory phase imaging. SI ratios can be used to confirm subtle internal enhancement and enhancement curves are predictive of RCC subtype. MR urography using conventional extracellular gadolinium must account for T2* effects; however, gadoxetic acid enhanced urography is an alternative. The purpose of this review it to highlight important technical and interpretive pearls and pitfalls encountered with MP-MRI of solid renal masses.

Influence of age, sex and rearing systems on Toll-like receptor 7 (TLR7) expression pattern in gut, lung and lymphoid tissues of indigenous ducks.

Abstract 1. The objective of the experiment was to determine the influence of age, sex and rearing system on Toll-like receptor 7 (TLR7) gene expression in gut, lung and lymphoid tissues and physiological responses to stress in male and female indigenous ducks of Tamil Nadu, India. 2. A total of 36 ducks (12 males and 24 females) were obtained from local farmers and tissue samples of gut tissues (duodenum, jejunum, ileum and caecum), lymphoid organs (spleen and bursa) and lungs were collected in RNAlater solution followed by RNA extraction. 3. After normalisation to ß-actin (endogenous control) qPCR analysis identified a significant effect of age, sex and rearing system on TLR7 expression in the ducks. 4. A significant up-regulation of TLR7 expression was observed in lungs, duodenum, jejunum, ileum and caecum of sexually mature (45 wk) compared with that of immature ducks (16 wk). Among sexes, male ducks had significantly higher TLR7 expression than female ducks. 5. Age and sex interactions were significant in lungs, duodenum, jejunum and caecum. Ducks reared in an extensive housing system showed significantly higher TLR7 expression in bursa, lungs, duodenum, ileum and caecum compared to intensively reared ducks. There were no effects of age, sex and rearing systems on TLR7 expression in the spleen. 6. The heterophil-to-lymphocyte ratio and serum corticosterone were higher in ducks reared on an intensive system compared with ducks from an extensive rearing system.

Synovial procollagenase activation by human mast cell tryptase dependence upon matrix metalloproteinase 3 activation.

Mast cells have been implicated in the pathogenesis of the matrix degradation observed in the cartilaginous and osseous structures of the rheumatoid joint. We previously reported that human mast cell tryptase, a 134-kD granule-associated neutral protease, is present in rheumatoid synovium and can activate collagenase in crude culture medium in vitro. the present study attempts to depict the precise mechanism of this activation. To express full activation of latent collagenase, matrix metalloproteinase 3 (MMP-3) or stromelysin, can be activated by tryptase in a time and dose-dependent manner. Tryptase was not capable of generating active collagenase in the crude media from cultured rheumatoid synoviocytes depleted of proMMP-3 by immunoadsorption. In addition, the function of the tissue inhibitor of metalloproteinases (TIMP) was not altered by tryptase, and SDS-PAGE analysis revealed no degradation of TIMP by tryptase. The tryptase dependent activation of synoviocyte procollagenase thereby appears to be entirely dependent upon its ability to activate proMMP-3.

Diversity of melanoma plasma membrane proteinases: inhibition of collagenolytic and cytolytic activities by minocycline.

The tissue-destructive proteinases of B16-BL6 melanoma cells from C57BL/6 mice and subcellular fractions were examined. Cancer cell organelles were isolated following nitrogen cavitation with the use of sucrose density gradient centrifugation. Serine, cysteine, and metalloproteinases were assayed with the use of radiolabeled proteins and synthetic substrates. Tumor-induced red blood cell lysis was quantitated by measurement of the release of isotope from 59Fe-labeled red blood cells (RBC) cocultivated with melanoma cells; the RBC were from Wistar rats. Enzyme inhibitors with specificity toward different classes of proteinases were used in the above assays to categorize the enzymes responsible for substrate degradation. Results indicated that intact melanoma cells, cell organelles, and cytosol contain proteinases that can degrade collagen and gelatin and lyse normal RBC. Melanoma plasma membranes are highly enriched in collagenase, gelatinase, cysteine proteinase, plasminogen activator, and cytolytic activity. The inhibition of tumor collagenolytic, gelatinolytic, and cytolytic activities by EDTA and 1,10-phenanthroline but not by diisopropyl fluorophosphate and N alpha-p-tosyl-L-lysine chloromethyl ketone indicates that metalloproteinases are the active enzymes in these assays. Minocycline, a synthetic tetracycline with demonstrable inhibitory activity with other mammalian collagenases, also inhibited melanoma collagenolytic and cytolytic activities.

Purification of a gelatin-degrading type IV collagenase secreted by ras oncogene-transformed fibroblasts.

Connective tissue matrix-degrading metalloproteinases play an important role in cancer invasion. In this report we describe the isolation of a metalloproteinase exhibiting both type IV collagenolytic and gelatinolytic activities from the conditioned medium of NIH-3T3 fibroblasts transformed with DNA containing an activated c-Harvey-ras oncogene from T24 bladder cancer cells. This tumor proteinase was purified by anion exchange chromatography, zinc-chelate Sepharose chromatography, and gel permeation chromatography. The final product was homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (relative molecular mass = 67,000). Gelatin zymography revealed two bands of gelatinolytic activity, corresponding to molecular weights of 67,000 and 62,000. Upon immunoblotting with the use of an affinity-purified polyclonal rabbit antibody to a peptide region of type IV collagenase that lacks homology with interstitial collagenase or stromelysin, the purified tumor enzyme was identified as type IV collagenase.

Enrichment of collagen and gelatin degrading activities in the plasma membranes of human cancer cells.

Interactions between connective tissue substrates and proteinases localized to the surface of cancer cells are implicated in cancer invasion. In this report we have compared the enrichment of collagen and gelatin degrading activities and cysteine proteinase(s) in well-characterized (enzyme markers and electron microscopy) subcellular membrane fractions isolated from human small cell lung cancer lines (NCI-H69 and NCI-H82) and the RWP-1 pancreatic cancer line. With each cell line collagenolytic, gelatinolytic, and cysteine proteinase activities were enriched 5- to 128-fold in the plasma membrane fractions with differences noted between microvilli versus smooth membrane profiles. Incubation of tumor plasma membranes with methyl-3H-labeled collagen resulted in extensive degradation of the gamma, beta, alpha 1, and alpha 2 chains, suggesting the combined action of metalloproteinases. Treatment of tumor plasma membranes with the chaotropic agent, 2 M KCl, did not diminish membrane collagen- or gelatin-degrading activity, but extensively leached out the cysteine proteinase, suggesting that the latter enzyme is not an integral membrane protein. Enzyme inhibitors specific for metalloproteinases and cysteine proteinase were used to corroborate enzymatic classification. In conclusion, we have demonstrated variations in the localization of proteinases in the plasma membrane domains of different human cancer cells.

Imaging in carpal instability.

Carpal instability is a complex and heterogeneous clinical condition. Management requires accurate identification of structural injury with an understanding of the resultant movement (kinematic) and load transfer (kinetic) failure. Static imaging techniques, such as plain film radiography, stress views, ultrasound, magnetic resonance, MR arthrography and computerized tomography arthrography, may accurately depict major wrist ligamentous injury. Dynamic ultrasound and videofluoroscopy may demonstrate dynamic instability and kinematic dysfunction. There is a growing evidence base for the diagnostic accuracy of these techniques in detecting intrinsic ligament tears, but there are limitations. Evidence of their efficacy and relevance in detection of non-dissociative carpal instability and extrinsic ligament tears is weak. Further research into the accuracy of existing imaging modalities is still required. Novel techniques, including four-dimensional computerized tomography and magnetic resonance, can evaluate both cross-sectional and functional carpal anatomy. This is a narrative review of level-III studies evaluating the role of imaging in carpal instability.

MRI-Guided Percutaneous Biopsy of Mediastinal Masses Using a Large Bore Magnet: Technical Feasibility.

OBJECTIVE: To evaluate the diagnostic accuracy and safety of magnetic resonance imaging (MRI)-guided percutaneous biopsy of mediastinal masses performed using a wide-bore high-field scanner. MATERIALS AND METHODS: This is a retrospective study of 16 consecutive patients (8 male, 8 female; mean age 74 years) who underwent MRI-guided core needle biopsy of a mediastinal mass between February 2010 and January 2014. Size and location of lesion, approach taken, time for needle placement, overall duration of procedure, and post-procedural complications were evaluated. Technical success rates and correlation with surgical pathology (where available) were assessed. RESULTS: Target lesions were located in the anterior (n = 13), middle (n = 2), and posterior mediastinum (n = 1), respectively. Mean size was 7.2 cm (range 3.6-11 cm). Average time for needle placement was 9.4 min (range 3-18 min); average duration of entire procedure was 42 min (range 27-62 min). 2-5 core samples were obtained from each lesion (mean 2.6). Technical success rate was 100%, with specimens successfully obtained in all 16 patients. There were no immediate complications. Histopathology revealed malignancy in 12 cases (4 of which were surgically confirmed), benign lesions in 3 cases (1 of which was false negative following surgical resection), and one inconclusive specimen (treated as inaccurate since repeat CT-guided biopsy demonstrated thymic hyperplasia). Sensitivity, specificity, positive predictive value, negative predictive value, and accuracy in our study were 92.3, 100, 100, 66.7, and 87.5%, respectively. CONCLUSION: MRI-guided mediastinal biopsy is a safe procedure with high diagnostic accuracy, which may offer a non-ionizing alternative to CT guidance.

The effect of experimental diabetes on the molecular characteristics of soluble rat-tail tendon collagen.

Acid soluble rat-tail tendon collagen was prepared from animals rendered diabetic by treatment with either streptozotocin or alloxan and from matched controls. In comparison to the normal, the diabetic collagens consistently demonstrated decreased solubility of reconstituted fibrils, marked increase in intrinsic viscosity and a decreased ratio of alpha to beta components. Electrophoresis in sodium dodecyl sulfate-polyacrylamide gels revealed a marked decrease in migration of alpha1, alpha2, and beta components from both types of diabetic collagen. These data indicate that diabetic collagens are larger than normal and are capable of higher degrees of polymerization due to increased intra- and inter-molecular interactions. These changes could explain, in part, the altered response of diabetic connective tissues to inflammation and trauma.

Diabetes stimulates procollagen degradation in rat tendon in vitro.

To identify the mechanisms responsible for the paucity of recently synthesized collagen in connective tissues during diabetes, in vitro procollagen metabolism was studied in non-diabetic (control) and diabetic rats. Achilles tendons from the two groups were incubated for 1-8 h (35 degrees C) in medium containing [14C]proline and the radiolabeled collagen in the tissue, and that released into the media, were examined by SDS-polyacrylamide gel electrophoresis and fluorography. The bulk of the radiolabeled collagen in tendon from the diabetics was recovered as degradation products; these, but also procollagen and collagen components, were prominent in the control tissues. Moreover, the collagenous components synthesized by the diabetic rat tendons were more readily digested in vitro by trypsin than those produced by control tissues. We conclude that diabetes reduces collagen accretion in connective tissues in part due to increased intracellular degradation of procollagen.

Metastatic mouse melanoma cells release collagen-gelatin degrading metalloproteinases as components of shed membrane vesicles.

The purpose of this study has been to compare collagen-gelatin degrading enzymes isolated from cancer cell organelles and cytosol to the metalloproteinases released by cancer cells. To this end, metastatic mouse melanoma cell organelles were isolated by sucrose density gradient centrifugation and metalloproteinases were assayed using native and denatured [methyl-3H]collagen substrates. Solubilized proteinases were purified by ammonium sulfate precipitation, anion exchange, concanavalin A affinity and gel-filtration column chromatographic procedures and characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The conclusions were as follows: malignant melanoma cells have a metalloproteinase (Mr = 59,000) which is shed from cells into conditioned medium as a component of intact membrane vesicles rather than as a soluble enzyme; storage of tumor-conditioned medium leads to the generation of autoactivated soluble metalloproteinases of lower molecular weight; purification of these metalloproteinase species yielded variant collagenases that have considerable gelatinolytic activity and a cleavage preference site for the Gly-Ile bond in a collagen-like synthetic octapeptide substrate which is typical for collagenase-type metalloproteinases. It is proposed that localization of potent proteinases to the surface of cancer cells facilitates the local breakdown of connective tissues during the invasive process.

A chemically modified tetracycline inhibits streptozotocin-induced diabetic depression of skin collagen synthesis and steady-state type I procollagen mRNA.

Wasting of connective tissues including skin, bone, and cartilage have been closely associated with elevated matrix metalloproteinase (MMP) activity and depressed collagen content in the streptozotocin (STZ)-induced diabetic rat, while tetracyclines have been reported to normalize total body weight, skin hydroxyproline and collagen content in this model, in part through inhibition of MMPs. In the present study, we report the effect of CMT-1, a chemically modified tetracycline that lacks antimicrobial properties but retains divalent cation binding and MMP inhibitory activity, on diabetic skin collagen synthesis and steady-state levels of procollagen alpha 1(I) mRNA. Male, 4-month old Sprague-Dawley rats received a single injection of 75 mg/kg STZ or citrate vehicle alone and diabetic status was confirmed by positive glucosuria. Some diabetic animals received 10 mg/day of CMT-1 by oral gavage and, 28 days after STZ treatment, body weight, blood glucose values and the in vivo rates of skin collagen production were measured using the pool-expansion technique. Steady-state levels of procollagen alpha 1(I) mRNA were analyzed 21 days after STZ treatment by hybridization of total RNA with a 32P labelled cDNA to rat type I procollagen alpha 1(I) mRNA in a dot-blot assay. STZ treatment was found to significantly depress body weight, skin collagen hydroxyproline content, the in vivo rate of collagen production, and hybridizable levels of type I procollagen alpha 1(I) mRNA. CMT-1 administered daily to STZ-treated rats inhibited the diabetic depression of these parameters but had little or no effect on non-diabetic controls or on STZ-induced hyperglycemia. Thus, in addition to the inhibition of MMP mediated extracellular collagen degradation, these results suggest CMT-1 also acts to inhibit diabetic connective tissue breakdown in STZ-induced diabetes by increasing both steady-state levels of type I procollagen mRNA and collagen synthesis through mechanism(s) that are independent of the antibacterial properties of tetracyclines.

Matrix metalloproteinase inhibitor prevents acute lung injury after cardiopulmonary bypass.

BACKGROUND: Acute lung injury (ALI) after cardiopulmonary bypass (CPB) results from sequential priming and activation of neutrophils. Activated neutrophils release neutral serine, elastase, and matrix metalloproteinases (MMPs) and oxygen radical species, which damage alveolar-capillary basement membranes and the extracellular matrix, resulting in an ALI clinically defined as adult respiratory distress syndrome (ARDS). We hypothesized that treatment with a potent MMP and elastase inhibitor, a chemically modified tetracycline (CMT-3), would prevent ALI in our sequential insult model of ALI after CPB. METHODS AND RESULTS: Anesthetized Yorkshire pigs were randomized to 1 of 5 groups: control (n=3); CPB (n=5), femoral-femoral hypothermic bypass for 1 hour; LPS (n=7), sham bypass followed by infusion of low-dose Escherichia coli lipopolysaccharide (LPS; 1 microgram/kg); CPB+LPS (n=6), both insults; and CPB+LPS+CMT-3 (n=5), both insults plus intravenous CMT-3 dosed to obtain a 25-micromol/L blood concentration. CPB+LPS caused severe lung injury, as demonstrated by a significant fall in PaO(2) and an increase in intrapulmonary shunt compared with all groups (P<0.05). These changes were associated with significant pulmonary infiltration of neutrophils and an increase in elastase and MMP-9 activity. CONCLUSIONS: All pathological changes typical of ALI after CPB were prevented by CMT-3. Prevention of lung dysfunction followed an attenuation of both elastase and MMP-2 activity. This study suggests that strategies to combat ARDS should target terminal neutrophil effectors.

Skin collagen metabolism in the streptozotocin-induced diabetic rat. Enhanced catabolism of collagen formed both before and during the diabetic state.

Collagen catabolism has been measured in skins of streptozotocin-induced diabetic rats. For measuring catabolism of collagen synthesized de novo during the diabetic state, we measured the amounts of [3H]hydroxyproline-containing degradation products in skins of diabetic rats, killed 4 h after [3H]proline injection (protocol 1); degradation products were isolated in TCA-soluble fractions of skin homogenates. For measuring catabolism of collagen preexisting before the induction of the diabetic state, we measured the 21-day loss of [3H]hydroxyproline (and hydroxyproline) in entire skins of rats that were streptozotocin-treated after [3H]proline injection (protocol 2). A 2.5-fold increase in the relative amounts of [3H]hydroxyproline-containing degradation products was measured in the TCA-soluble fractions of skins from diabetic rats (protocol 1). These degradation products had a low molecular weight (as evident from their diffusibility), and they were derived from recently synthesized collagen, possibly procollagen (as evident from their high [3H]hydroxyproline specific activity). Furthermore, they were not derived from the degradation of [3H]hydroxyproline-labeled collagen present before induction of the diabetic state (protocol 2). Evidence for this conclusion is as follows: the amounts of [3H]hydroxyproline-containing degradation products in skins of diabetic rats were not greater than that in skins of control rats, despite a 50% resorption of collagen in skins of diabetic rats. Overall, the catabolism of collagen formed de novo during the diabetic state was distinguished from the catabolism of collagen formed before, and both catabolic processes were enhanced in rat skins of streptozotocin-induced diabetic rats.

Reactive oxygen species activate and tetracyclines inhibit rat osteoblast collagenase.

Recent studies have demonstrated that tetracyclines (TCs) scavenge reactive oxygen species (ROS). Hypochlorous acid (HOCl), an ROS produced by neutrophils, has been shown to activate neutrophil procollagenase. The objective of the present study was to determine whether (1) HOCl also activated osteoblast procollagenase and (2) TCs inhibited this enzyme in the presence of HOCl. HOCl (5 microM) activated the proenzyme approximately sixfold (P < 0.01) from the medium of PTH-treated UMR-106-01 osteoblastic osteosarcoma cells as determined by functional collagenase assay (3H-methyl-labeled collagen substrate). Doxycycline (50-400 microM) and chemically modified tetracycline, CMT-1 (100-400 microM), significantly inhibited collagenase activity 50-90% and 40-80%, respectively, in the presence of 5 microM HOCl. Concentrations of 6-25 microM doxycycline and 10-50 microM CMT-1 had no significant effect. Furthermore, an excess concentration of cation (50 mM CaCl2 or 50 microM ZnCl2) added to the incubation mixtures containing either doxycycline or CMT-1 did not restore collagenase activity, as demonstrated by SDS-PAGE-fluorography. These data suggested that TCs reduced available HOCl and thus prevented the hypochlorous acid conversion of the osteoblast proenzyme to active collagenase. TCs may have therapeutic potential in the treatment of periodontitis and other diseases by several mechanisms that inhibit pathologic collagen breakdown.

Biodistribution of radiolabeled [(3)H] CMT-3 in rats.

CMT-3 is a NON-ANTIMICROBIAL tetracycline (TC), chemically modified to enhance its collagenase-inhibitory property. This property is therapeutically useful in treating diseases such as periodontitis, cancer and arthritis. CMT-3 was labeled with tritium [(3)H] at Carbon 7. Four adult male Sprague-Dawley rats (350--400 g body weight) were gavaged once with a mixture of cold CMT-3 and [(3)H] CMT-3 (750 microCi). An additional four rats were gavaged for 2 days with cold CMT-3(15 mg/Kg/day) and on the third day the rats were gavaged with a mixture of cold and [(2)H] CMT-3 (750 microCi); and all 8 rats were placed in the metabolic cages. Blood samples were collected from the tail at multiple intervals from 1--14 hr after [(3)H] CMT-3 administration. At 14 hr, the rats were anesthetized, euthanized and various tissues including visceral organs were removed and weighed. The contents of GI tracts were emptied and added to the fecal pellets and weighed. The urine samples were collected and volume measured. Each tissue or organ was minced finely with scissors and 100 mg of tissue was digested in 1 ml of Tissue-solv (Packard Lab), for 4 hrs at 37 degrees C and each sample was diluted up to 10 ml of distilled water. A 100 microl aliquot was taken and diluted with an equal volume of glacial acetic acid, 10 ml of Atom-lite was added and counted for radioactivity in a liquid scintillation spectrometer. This biodistribution study revealed that over 14 hrs, 54% and 3% of [(3)H] CMT-3 were excreted in the feces and urine, respectively. The serum [(3)H] CMT-3 count reached its maximum value at about 12 hours. The tissues retained the CMTs as follow: muscle (23%); skin (2.41%); bone (1.72%); and the brain retained 0.21% of the label. The radioactive CMT-3 in the visceral organs is as follows: GI tract - its contents (8.9%); heart (0.41%), testis (0.41%); lungs >(0.16%); spleen (0.08%); liver (0.03%); kidneys > (0.02%).

Excessive matrix metalloproteinase activity in diabetes: inhibition by tetracycline analogues with zinc reactivity.

Diabetes mellitus in rats is characterized by excessive activity of several matrix metalloproteinases (MMPs), notably collagenase(s) and gelatinase(s), in skin, gingiva, and other tissues. A number of tetracyclines (TCs), both antimicrobial compounds as well as chemically modified non-antimicrobial TC analogues (CMTs) are known to possess potent inhibitory activity against these enzymes. Three conventional antimicrobial TCs and six CMTs were used in this study. In vitro, doxycycline was shown to possess higher inhibitory capacity (i.e. lower IC(max)) against diabetic rat skin collagenase than either minocycline or tetracycline HCl. Addition of excess zinc partially reversed the proteinase inhibition by TCs. In vivo, using rats made diabetic with streptozotocin (STZ), oral administration of various TCs led to decreased weight loss and substantial reductions in the activity of both skin collagenase and skin gelatinase (primarily MMP-9, 92 kDa) without affecting blood glucose. Using an in vitro spectrophotometric technique, the Zn(++) reactivity of several CMTs was assessed and found to be positively related to the potency of these compounds as MMP inhibitors. One particular CMT (CMT-5, pyrazole analogue), which is neither antimicrobial nor capable of binding metal cations, did not inhibit the MMPs. TCs have potential utility in management of diabetic complications mediated by excessive activity of MMPs.

The lipophilicity, pharmacokinetics, and cellular uptake of different chemically-modified tetracyclines (CMTs).

CMTs are analogs of tetracyclines, which are chemically modified to eliminate their antimicrobial efficacy but which retain their inhibitory activity against matrix metalloproteinases. These compounds have been found to inhibit connective tissue breakdown in animal models of diseases such as periodontitis, arthritis and cancer. Because CMTs exhibit different in vivo efficacy in these various models of disease, the current study compared their pharmacokinetics and other properties as follows: Adult male Sprague-Dawley rats were administered by oral gavage a single dose of 5mg of different CMTs suspended in 1 ml 2% carboxymethyl-cellulose, and blood samples were collected from 1-48 hours after dosing. The sera were extracted, then analyzed by HPLC using a C-18 reverse-phase column. The results showed that the peak concentrations (C(max)) in rat sera 1-12 hours after oral administration of CMTs -1, -2,-3, -4,-5,-6,-7,-8 and doxycycline were 5.5, 0.7, 4.6, 6.2, 0.8, 0.7, 9.0 (note: the 3 peaks detected for CMT-7 were combined), 15.0 and 0.9 microg/ml, respectively. Their in vivo half-lives (t(1/2)) were 11, 5, 22, 11, 32, 15, 37, 38, and 17 hours, respectively. Of the anticollagenase CMTs tested, CMT-8 showed the greatest C(max) and t(1/2)values, followed by CMTs-3, -1, -4, and perhaps -7; CMTs-2, -5, and -6 exhibited much lower levels in serum. The relative lipophilicities of the 8 CMTs and doxycycline were tested by examining their extractability in octanol. The results showed that CMT-2, -5, and -6 had the lowest partition coefficients using this organic solvent, while CMT-3 was the most lipophilic. The lipophilicity of the different CMTs was also positively correlated (r(2)=0.767, P<0.05) to peak serum concentrations (C(max)), but not to their serum half-lives (r(2)=0.25,P=0.49). This property of the different CMTs was also found to be positively correlated to their ability to enter into human whole blood cells in vitro (r2=0.95, P<0.001). Since CMT-8, as well as CMTs-3 and -1, consistently exhibited the greatest in vivo efficacy in animal models of tissue breakdown, this may reflect, at least in part, their favorable pharmacokinetics and tissue uptake.

Wound healing in ovariectomized rats: effects of chemically modified tetracycline (CMT-8) and estrogen on matrix metalloproteinases -8, -13 and type I collagen expression.

Cutaneous wound healing is a complex process involving interactions of various cell types. Skin, in addition to certain other organs, is dependent on estrogen; and estrogen-deficiency is associated with impaired wound healing. Wound healing involves the action of collagenolytic matrix metalloproteinases (MMPs). We investigated the expression and localization of collagenolytic MMPs -8 and -13 by collagenase activity assay, Western immunoblot analysis, in situ hybridization and immunohistochemical staining as well as type I collagen by hydroxyproline content analysis and immunohistochemical staining in cutaneous wounds from aged Sham and ovarioectomized (OVX) rats. After wounding, OVX rats were treated with either placebo, chemically modified tetracycline-8 (CMT-8) or estrogen. We found that MMP-8 and MMP-13 mRNA were expressed in wound epithelium of all samples examined as evidenced by in situ hybridization. Type I collagen, which was abundant in all groups examined, was decreased in OVX rats, but was increased by both CMT-8 and estrogen treatments to the level of Sham group. Hydroxyproline analysis revealed similar results. Western blot data showed that all forms of MMP-8 and MMP-13 were clearly reduced in the CMT-8 treated group compared to OVX. Analysis of collagenolytic activity confirmed the decreased collagenolysis in skin wound extracts from CMT-treated rats when compared with skin wound extracts from OVX rats. Our results show for the first time that MMP-8 mRNA and protein are expressed in rat wound epithelium. We further show that CMT-8 and estrogen have a beneficial effect on skin wound healing in OVX rats by increasing the collagen content and reducing the MMP-mediated collagenolysis.

A combination of subtherapeutic doses of chemically modified doxycycline (CMT-8) and a bisphosphonate (clodronate) inhibits bone loss in the ovariectomized rat: a dynamic histomorphometric and gene expression study.

Recent studies have demonstrated that tetracyclines can reduce bone loss in the ovariectomized (OVX) rat model of osteoporosis. In the current study, a non-antimicrobial, chemically modified doxycycline (CMT-8), alone or in combination with a bisphosphonate (Clodronate), was evaluated in this model. Forty-two, 6month old, female rats were randomly assigned to the following groups, (6/ group): a) sham/vehicle, b) OVX/vehicle; c) OVX/1 mg/day CMT-8; d) OVX/2 mg/day CMT-8, e) OVX/1 mg/week Clodronate; and f) OVX/1 mg/day CMT-8 + 1 mg/week Clodronate, CMT-8 was administered by oral gavage, Clodronate injected S/C. Following sham surgery or OVX, the rats were treated for 90 days with CMT-8 or vehicle alone, injected at three different times with fluorochrome labels, the rats were sacrificed, and the tibiae excised for analysis by dynamic bone histomorphometry. Femurs were aseptically removed and analyzed for collagen, collagenase and osteopontin mRNAs by Northern and dot blot analysis. As expected, OVX decreased trabecular bone volume (BV/TV by 73.8% vs. sham p<.01), and also reduced trabecular thickness, numbers, and increased spacing. Bone loss in the OVX animals was partially prevented with either 2 mg/day CMT-8 or 1 mg/wk Clodronate (p<.01), while the 1 mg/day CMT-8 had no effect. Interestingly, the efficacy of the combination therapy of CMT-8 and Clodronate was significantly better than either treatment by itself, maintaining bone mass and structural indices at levels identical to sham values. OVX rats mRNA for collagen, collagenase and osteopontin were elevated indicating high-turnover bone loss. Only COMBO therapy significantly reduced the collagenase and osteopontin mRNA. In summary, CMT-8 mono-therapy (2 mg) alone partially inhibited bone loss in this animal model of osteoporosis. However, 1 mg/day (CMT-8) monotherapy had no effect on bone loss or bone mRNA levels and when combined with Clodronate, interacted to increase efficacy. Thus, a combination of a suboptimal dose of CMT-8 and a bisphosphonate appears to increase the amount of bone by suppressing resorption in a model of osteoporosis.