Exploring Canine-Human Differences in Product Performance. Part II: Use of Modeling and Simulation to Explore the Impact of Formulation on Ciprofloxacin In Vivo Absorption and Dissolution in Dogs.

This study explored the in vivo performance of three oral ciprofloxacin formulations (oral solution, fast, or slow dissolving tablets) in beagle dogs. The in vivo absorption and dissolution behaviors, estimated with in silico mechanistic models, were compared to the results previously published in human volunteers. Six normal healthy male beagle dogs (five to completion) received three oral formulations and an intravenous infusion in a randomized crossover design. Plasma ciprofloxacin concentrations were estimated by tandem mass spectrometry detection. A mechanistic absorption model was used to predict the in vivo dissolution and absorption characteristics of the oral formulations. Canine ciprofloxacin absorption was constrained to the duodenum/jejunum. This absorption window was far narrower than that seen in humans. Furthermore, while substantial within-individual variability in drug absorption was seen in human subjects, a greater magnitude of variability was observed in dogs. For three sets of data, a lag time in gastric emptying was necessary to improve the accuracy of model-generated in vivo blood level profile predictions. In addition to species-associated dissimilarities in drug solubilization due to human versus canine differences in gastrointestinal fluid compositions, the far more rapid intestinal transit time and potential segmental differences in drug absorption needed to be considered during human-canine extrapolation of oral drug and drug product performance. Through the use of mechanistic models, the data generated in the human and canine studies contributed insights into some aspects of the interspecies differences to be considered when extrapolating oral bioavailability/formulation effect data between dogs and humans.

Review of global regulations concerning biowaivers for immediate release solid oral dosage forms.

The regulations with respect to biowaivers for immediate release (IR) solid oral dosage forms in the USA, the EU, Japan and from the World Health Organization (WHO) are summarized and compared. Two case studies are presented, one from our own files and one from the open literature, showing the similarities and the differences among the qualification requirements of the four systems. The regulatory experience gained up to now is reviewed and expected future trends are discussed.

Collection and preparation of blood products.

Blood-component therapy has become an integral part of veterinary practice. Although access to veterinary blood banks has increased, practitioners may prefer to create their own blood-donor program to provide for their blood-product needs or to respond to an emergent need. Before embarking on such an endeavor, it is important to understand the techniques and requirements for such a program. This article will discuss issues in donor selection and management, supplies and techniques of blood-component acquisition, and supplies and techniques of blood-component preparation.

Blood pressure effects of thoracic gunshot wounds: the role of bullet image diameter.

Differences in handgun bullet diameter, expansion, and penetration (no exit) versus perforation (with exit) may be the cause of variable blood pressure effects after thoracopulmonary injury. Forty nonlethal isolated gunshot wounds of the thorax were evaluated excluding wounds of the heart, great vessels, and spinal cord. Chest radiographs were assessed for bullet base diameter, bullet expansion, and wound length. Large bullets were defined as having radiographic base images of 9 mm or more in diameter. Systolic blood pressures were compared between penetrating large and small bullet groups and with perforating wounds. Response times and demographics were compared. Wounds caused by large bullet penetration resulted in lower initial systolic blood pressures than wounds caused by small bullet penetration (98 vs 125 mm Hg, P < 0.05). The average age, transport time, and wound length were similar among the bullet groups. We conclude that penetrating thoracopulmonary wounds caused by large bullets resulted in lower initial systolic blood pressure.

Development of a more rapid, reduced serum culture system for Caco-2 monolayers and application to the biopharmaceutics classification system.

The objectives were: (1) to develop a more rapid, reduced serum culture system for Caco-2 monolayers, relative to the traditional 21-day, 10% fetal bovine serum (FBS) system; and (2) to determine the biopharmaceutical drug classification of an oral therapeutic agent using this new system. Caco-2 cells were grown in the six well format on polycarbonate filters, in medium containing 2% iron supplemented calf serum (sCS) and a combination of growth factors and hormones. After 4 days in culture, permeabilities of three marker compounds (metoprolol, mannitol, and taurocholate) across monolayers were determined, and compared to permeabilities from the traditional 21-day, 10% FBS system, using cells at similar passage number. Cell morphology, degree of cell differentiation, and the presence of two efflux pumps were assessed. The 2% sCS model was also used to classify the permeability of an oral therapeutic agent as high or low. No difference in permeability was observed for metoprolol transport (P = 0.38) between the two culture methods, and the values obtained were independent of passage number of the cells. Mannitol permeability was about 2-fold higher from the 2% sCS system, as compared to the 10% FBS system. Taurocholate permeability was low indicating the 2% sCS culture at 4 days lacked this particular active transporter capability. Electron micrographs of cells grown in the 2% sCS system at 4 days revealed the presence of microvilli and tight junctions. P-glycoprotein and an efflux pump for furosemide were functionally present. The 2% sCS system indicated the oral therapeutic agent as highly permeable, which agreed with the 10% FBS system. This new system provides a rapid, accurate, and economical option for passive permeability determination, and appears to be applicable to the proposed Biopharmaceutics Classification System (BCS).

Influence of passive permeability on apparent P-glycoprotein kinetics.

PURPOSE: The objectives of this work were to evaluate the importance of moderate passive permeability on apparent P-glycoprotein (P-gp) kinetics, and demonstrate that inspection of basolateral to apical and apical to basolateral (BL-AP/AP-BL) permeability ratios may result in a compound being overlooked as a P-gp substrate and inhibitor of another drug's transport via P-gp inhibition. METHODS: The permeability ratios of nicardipine, vinblastine, cimetidine, and ranitidine were determined across Caco-2 monolayers that express P-gp, in the presence and absence of the specific P-gp inhibitor, GF120918. In addition, the permeability ratio of vinblastine was studied after pretreatment of Caco-2 monolayers with nicardipine, ranitidine, or cimetidine. Similar studies were repeated with hMDRI-MDCK monolayers. RESULTS: The permeability ratios for cimetidine and vinblastine were >2. The permeability ratios for nicardipine and ranitidine were close to unity, and were not affected by the addition of GF120918. Based solely on ratios, only compounds with moderate transcellular permeability (vinblastine and cimetidine) would be identified as P-gp substrates. Although the permeability ratios appeared to be unity for nicardipine and ranitidine, both compounds affected the permeability of vinblastine, and were identified as substrates and inhibitors of P-gp. Studies performed in hMDR1-MDCK cells confirmed these experimental results. Data were explained in the context of a kinetic model, where passive permeability and P-gp efflux contribute to overall drug transport. CONCLUSIONS: Moderate passive permeability was necessary for P-gp to reduce the AP-BL drug permeability. Inspection of the permeability ratio after directional transport studies did not effectively identify P-gp substrates that affected the P-gp kinetics of vinblastine. Because of the role of passive permeability, drug interaction studies with known P-gp substrates, rather than directional permeability studies, are needed to elucidate a more complete understanding of P-gp kinetics.

Structure of poliovirus type 2 Lansing complexed with antiviral agent SCH48973: comparison of the structural and biological properties of three poliovirus serotypes.

BACKGROUND: Polioviruses are human pathogens and the causative agents of poliomyelitis. Polioviruses are icosahedral single-stranded RNA viruses, which belong to the picornavirus family, and occur as three distinct serotypes. All three serotypes of poliovirus can infect primates, but only type 2 can infect mice. The crystal structures of a type 1 and a type 3 poliovirus are already known. Structural studies of poliovirus type 2 Lansing (PV2L) were initiated to try to enhance our understanding of the differences in host range specificity, antigenicity and receptor binding among the three serotypes of poliovirus. RESULTS: The crystal structure of the mouse neurovirulent PV2L complexed with a potent antiviral agent, SCH48973, was determined at 2.9 A resolution. Structural differences among the three poliovirus serotypes occur primarily in the loop regions of the viral coat proteins (VPs), most notably in the loops of VP1 that cluster near the fivefold axes of the capsid, where the BC loop of PV2L is disordered. Unlike other known structures of enteroviruses, the entire polypeptide chain of PV2L VP4 is visible in the electron density and RNA bases are observed stacking with conserved aromatic residues (Tyr4020 and Phe4046) of VP4. The broad-spectrum antiviral agent SCH48973 is observed binding in a pocket within the beta-barrel of VP1, in approximately the same location that natural 'pocket factors' bind to polioviruses. SCH48973 forms predominantly hydrophobic interactions with the pocket residues. CONCLUSIONS: Some of the conformational changes required for infectivity and involved in the control of capsid stability and neurovirulence in mice may occur in the vicinity of the fivefold axis of the poliovirus, where there are significant structural differences among the three poliovirus serotypes in the surface exposed loops of VP1 (BC, DE, and HI). A surface depression is located at the fivefold axis of PV2L that is not present in the other two poliovirus serotypes. The observed interaction of RNA with VP4 supports the observation that loss of VP4 ultimately leads to the loss of viral RNA. A model is proposed that suggests dual involvement of the virion fivefold and pseudo-threefold axes in receptor-mediated initiation of infection by picornaviruses.

A simple device for closure of fasciotomy wounds.

Early decompressive fasciotomy is essential in the prevention of the sequelae of compartment syndrome. Many techniques have been described for the closure of the fasciotomy wound, and controversy exists as to which method is the best. Primary closure is often impossible secondary to tissue retraction and edema. Split-thickness skin grafting leaves a thin, insensate, and often aesthetically unpleasing result. Gradual mechanical dermal apposition has been used with increasing frequency, and has been shown to be effective in the closure of fasciotomies, but often takes 7-10 days for closure. We present our experience with the STAR, a mechanical method of fasciotomy wound closure that is effective in 2-4 days, and is extremely simple to use.

Evaluating blunt abdominal trauma:role for ultrasonography.

Patients in unstable condition with suspected blunt abdominal trauma require rapid evaluation to assess the need for laparotomy. Ultrasonography is fast and uses portable equipment, and it can be used in the resuscitation area to detect free intraperitoneal fluid as an indication of intraabdominal injury in the patient in unstable condition. To determine the utility of emergency abdominal sonography for evaluating patients with blunt abdominal trauma, a prospective study was designed to compare ultrasonography to diagnostic peritoneal lavage. Emergency sonography was performed prior to diagnostic peritoneal lavage on 54 acutely injured patients in the resuscitation area. Our results reveal that ultrasonography has a sensitivity of 87%, a specificity of 100%, and an overall accuracy of 96% for detecting free intraperitoneal fluid. We conclude that ultrasonography is a reliable method for the emergent evaluation of blunt abdominal trauma and can be used in place of diagnostic peritoneal lavage as the initial indicator of significant intraperitoneal injury requiring surgical intervention.

1,000 consecutive ultrasounds for blunt abdominal trauma.

Diagnostic peritoneal lavage (DPL) and computed tomography (CT) are the primary diagnostic modalities used in the evaluation of patients with suspected blunt abdominal trauma (BAT). DPL is fast and accurate but is associated with complications. CT is also accurate, yet requires stability and transportability of the patients. Ultrasound (US) has been suggested as an aid in evaluating BAT. We evaluated US in the initial assessment of BAT in 1000 patients. Patients were eligible for the study if they met specified trauma criteria and had suspected BAT. We then followed the outcome of the patients and their further work-up. US showed a sensitivity of 88%, a specificity of 99%, and an accuracy of 97% for detecting intraabdominal injuries. We conclude that emergency ultrasound may be used as the initial diagnostic modality for suspected blunt abdominal trauma.

First place winner of the Conrad Jobst Award in the gold medal paper competition. Nitric oxide synthase inhibitors N-monomethylarginine and aminoguanidine prevent the progressive and severe hypotension associated with a rat model of pancreatitis.

The objective of this study was to determine whether the observed vascular collapse and other pathologic features of severe pancreatitis may be related to the induction of nitric oxide synthase (NOS). The rat model of pancreatitis reported by Schmidt et al. was employed. Rats in the experimental groups received pretreatment with known NOS inhibitors, N-Monomethylarginine (NMMA) or Aminoguanidine (AG). Controls included sham-operated rats without pancreatic insult and a diseased control group which received pretreatment with normal saline (NS). Arterial blood pressure was continuously recorded with a femoral arterial catheter connected to a transducer and monitor. Fluid resuscitation for hypotension followed a strict protocol with the administration of 5.0 cc NS for sustained decreases in systolic blood pressure (SBP) below 90 mm Hg at 5-minute intervals. Laboratory parameters and histopathology confirmed the induction of pancreatitis, with 6 to 15-fold increases in serum amylase levels and an average of approximately 20% decrease in serum ionized Ca++ levels. Immunohistochemical studies of the pancreas revealed that pancreatic insult resulted in the induction of NOS. Rats in the saline control group (n = 5) became hypotensive (SBP less than 90 mm Hg) between 3 and 4 hours post pancreatic insult and required an average of 110.0 cc (3-4 x blood volume) of NS fluid resuscitation. Rats which were not resuscitated (n = 5) did not survive.(ABSTRACT TRUNCATED AT 250 WORDS)

Can ultrasound replace diagnostic peritoneal lavage in the assessment of blunt trauma?

Diagnostic peritoneal lavage (DPL) and computed tomography (CT) are the primary diagnostic modalities in the evaluation of patients with suspected blunt abdominal trauma (BAT). Diagnostic peritoneal lavage is fast and accurate but associated with complications. Computed tomography is also accurate, yet requires that patients be stable and transportable. A prospective study was designed to determine the utility of emergency ultrasound (US) studies in the initial assessment of BAT. Two hundred acutely injured patients with suspected BAT were evaluated with US. Patients were eligible for the study if they met trauma criteria and had suspected BAT. Subsequently, without knowledge of the US results, DPL or CT was performed. Ultrasound showed a sensitivity of 83%, a specificity of 100%, and an accuracy of 97% in detecting intra-abdominal injuries. Six injuries were missed but only one was felt to be significant. If US had been used in all 200 patients, 199 would have had appropriate care. We conclude US is reliable in the detection of free intraperitoneal fluid and may be used in place of DPL or CT.

Fears and worries of young children as expressed in a contextual play setting.

One hundred 5- and 6-year-old children were studied with a contextual play technique in order to gain insight into their fears and worries. Sex differences and differences relating to questioning procedure were examined. The discussion included the potential usefulness of the method and some implications for parents and teachers.

A new mechanism in one-kidney, one clip hypertension.

The renin-angiotensin system does not appear to be involved in the maintenance of elevated blood pressure in experimental one-kidney, one clip hypertension. Paradoxically, direct immunization with purified hog kidney renin lowers the blood pressure of rabbits with this form of hypertension. Antirenin antibodies were removed and the IgG fraction prepared from the plasma of such immunized rabbits. The antibodies thus obtained lowered the blood pressure of other hypertensive rabbits. The same antibodies, detected with a fluorescein-labeled second antibody, stained the cytoplasm of smooth muscle and certain other cells in sections of kidney, aorta, carotid artery, heart, liver, pancreas, adrenal gland, and small intestine from normal and hypertensive rabbits. We suggest that renin is converted into a form that is present most conspicuously in arterial and arteriolar smooth muscle. Its function in this location is unknown but must involve vasoconstriction as its neutralization by specific antibody lowers the blood pressure of one-kidney, one clip hypertensive rabbits.

Purification of hog kidney renin with immobilized monoclonal antirenin.

Spleen cells from mice immunized with partially purified hog kidney renin were fused with mouse myeloma cells to produce a stable monoclonal hybridoma cell line that synthesizes an antibody against renin. A single monoclonal antibody was chosen for study and has been produced in large quantity and purified by affinity chromatography on protein A-Sepharose. The antirenin, which belongs to the IgG1 subclass, exhibits anticatalytic activity against both hog and rabbit renin. An immunoaffinity column prepared from antibody coupled to Sepharose has been used in the purification of renin from hog kidney. Although renin is quantitatively adsorbed from solution, it can be eluted from the column under gentle conditions. The highly purified renin, with specific activity of 2122 Goldblatt Units/mg protein, exhibits both charge (pH 4.1 to 5.1) and size (38,000 to 42,700) heterogeneity. Hog kidney renin dissociates in the presence of sodium dodecyl sulfate (SDS) and mercaptoethanol to heavy and light chains with molecular weights of 33,700 and 5,800, respectively. In the presence of SDS, a small amount of a nw form of renin is observed with a molecular weight of 19,500 which retains activity on renaturation. The monoclonal antibody should be a useful tool for the study of the renin-angiotensin system and especially for the purification of renin. The hybridoma cell line used in this study (F-32 VIII C4) has been donated to the American Type Culture Collection.

Uveitis caused by cytotoxic immune response to cutaneous malignant melanoma in swine: destruction of uveal melanocytes during tumor regression.

In a substrain of Sinclair miniature black swine, bred for increasing incidence of cutaneous malignant melanomas, tumor regression occurs spontaneously and is accompanied by depigmentation of the skin, hair, and eyes. We conducted a 12-month longitudinal study of the ocular phenomena in 30 swine beginning at 3 weeks of age. The clinically observed sequence of depigmentation of the fundus and iris was correlated with histopathologic changes in selected enucleated eyes. Normal melanocytes of the uveal tract are destroyed between the 4th and 16th week of life. Melanocyte destruction is preceded by an invasion of the uveal tract by mononuclear cells having the ultrastructural features of lymphocytes and monocytes. Melanin and other cellular debris of ruptured melanocytes are ingested by macrophages which then migrate to the walls of blood vessels. Cataracts and band keratopathy develop secondary to the uveitis in some animals. Pilot electroretinograms show diminished electrical activity in photoreceptors of totally depigmented eyes possibly indicating ischemic or toxic damage to the retina. The retinal pigment epithelium remains essentially normal during the acute stages of uveal inflammation; later some damage and reparative hyperplasia may occur. The death of normal uveal melanocytes that occurs during the systemic attack on the cutaneous malignant melanomas appears to be an "innocent bystander" error in the immune recognition mechanism. The antigenic basis of this immunologic cross reaction is under investigation.