WO1999061058A1 - Method of altering heartbeat - Google Patents

Method of altering heartbeat Download PDF

Info

Publication number
WO1999061058A1
WO1999061058A1 PCT/GB1999/001418 GB9901418W WO9961058A1 WO 1999061058 A1 WO1999061058 A1 WO 1999061058A1 GB 9901418 W GB9901418 W GB 9901418W WO 9961058 A1 WO9961058 A1 WO 9961058A1
Authority
WO
WIPO (PCT)
Prior art keywords
microcapsules
ventricular
infusion
patient
pvc
Prior art date
Application number
PCT/GB1999/001418
Other languages
French (fr)
Inventor
Paulus Antonius Van Der Wouw
Original Assignee
Quadrant Healthcare (Uk) Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Quadrant Healthcare (Uk) Limited filed Critical Quadrant Healthcare (Uk) Limited
Priority to AU39382/99A priority Critical patent/AU3938299A/en
Publication of WO1999061058A1 publication Critical patent/WO1999061058A1/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/22—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations
    • A61K49/222—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations characterised by a special physical form, e.g. emulsions, liposomes
    • A61K49/223—Microbubbles, hollow microspheres, free gas bubbles, gas microspheres
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0028—Disruption, e.g. by heat or ultrasounds, sonophysical or sonochemical activation, e.g. thermosensitive or heat-sensitive liposomes, disruption of calculi with a medicinal preparation and ultrasounds

Definitions

  • the present invention relates to a method of inducing a ventricular premature heart beat in a human or animal patient. Such induction may be beneficial in the medical field, in relation to the diagnosis of coronary arrhythmias.
  • the method utilizes the introduction into the patient of hollow microcapsules, and their interaction with ultrasound.
  • Air bubbles are known in the art for use in echocardiography techniques, in which a combination of hollow microcapsules intravenously injected into the body and ultrasound techniques are used to investigate the heart.
  • WO 92/18164 discloses the spray drying of a solution of a wall forming material, preferably a protein such as albumin, to form microcapsules.
  • a method for forming microcapsules which can be used as an echogenic contrast agent, which involves providing a solution of a material (which is advantageously a protein) in a solvent which comprises water and a liquid of greater volatility than water, and spraying the solution into a gas to evaporate the aqueous solvent, thereby providing hollow microcapsules.
  • microcapsules of the type described in WO 96/15814 in echocardiography techniques we have surprisingly found that the administering of such microcapsules can induce in patients a ventricular premature beat.
  • the monitoring and observation of such a ventricular premature beat can provide useful information on the patient, and in particular can be used to investigate ventricular tachycardia, and identify the source of an ectopic beat.
  • Such a method represents a highly advantageous advance on other diagnostic methods for ventricular tachycardia, which currently induce ventricular premature beats either by invasive techniques including catheter placement, or by gross electrical stimulation to the thorax.
  • the stimulation of ventricular premature beats in patients can be used to identify arrhythmias, and the type and origin of arrhythmia involved. They can also be used to determine whether anti-arrhythmic medication administered to patients who have previously experienced arrhythmias, ventricular tachycardia or ventricular fibrillation has been successful.
  • the method of the invention may also provide a benefit in the testing of automatic implantable cardioverter/defibrillator devices (AICD).
  • AICD automatic implantable cardioverter/defibrillator devices
  • Ventricular premature beats are also known as premature ventricular contractions or ventricular extrasystoles, and are heartbeats where the electrical origin or pacemaker is within the ventricular myocardium, as opposed to a sinus beat or supraventricular beat, where the electrical origin lies within the atrial myocardium. Such a beat also falls prior to when the next beat in a regular heart rhythm is expected, in contrast to an escape beat.
  • ventricular premature beats appear prematurely during regular sinus rhythm or any other non-ventricular rhythm, and are characterised by broad QRS-complexes ( >0.11s) without a preceding p-wave, or more precisely, without a preceding His bundel signal, which cannot normally be observed on a 12-channel electrocardiograph.
  • Such beats occur in normal healthy hearts, as well as under pathological conditions. In the latter case, they may precede or induce ventricular tachycardia. In particular in ischemic hearts, this may lead to life threatening situations.
  • a method of inducing in a human or animal patient a ventricular premature beat comprising intravenously administering thereto a composition containing hollow microcapsules, and exposing the patient to ultrasound.
  • hollow microcapsules for the manufacture of a diagnostic agent for the induction of ventricular premature beats in a patient.
  • a method of inducing and detecting in a human or animal patient a ventricular premature beat comprising intravenously administering thereto a hollow microcapsule, exposing the patient to ultrasound, and examining the patient to detect the presence or otherwise of a ventricular premature beat.
  • ventricular premature beats can be observed in the patient using electrocardiograph techniques, in particular conventional 12-channel EKG or body-surface EKG are preferred to detect the presence of the ventricular premature beat, or the induction of ventricular tachycardia.
  • the method of the invention utilizes ultrasound which conveniently has a Mechanical Index of 1.5, or above, conveniently 1.5, and also systolic triggering in order to significantly increase the number of ventricular premature beats in patients.
  • Mechanical Index is the measure of the ultrasound output of a suitable ultrasound device, and is defined as the maximal acoustic pressure in MPa, normally at the focal point, divided by the square root of the ultrasound frequency in MHz.
  • the method according to the invention may preferably have utility in a substantial proportion of the population - ie it will induce ventricular premature beats in at least 0.5 % , preferably at least 0.7% , more preferably at least 1 % of the population.
  • the method according to the invention may also conveniently be used as a step in a diagnostic method for the types of condition described above.
  • the method of the invention may be used to induce ventricular premature beats at specific QRS to ventricular premature beat intervals.
  • the QRS to ventricular premature beat interval can be changed by changing the QRS to trigger interval; a ventricular premature beat will occur approximately 80ms after the trigger.
  • a preferred microcapsule for use in the method of the invention is that described in WO 96/15814.
  • the preferred microcapsules thus are proteinaceous, have a relatively narrow size distribution and thinner shells, and can be made by spray drying an aqueous solution of protein, with the solvent comprising a solvent more volatile than water.
  • suitable volatile liquids include ethanol (a preferred volatile liquid; boiling point 78.3°C), methanol (boiling point 64.5°C) and acetone (boiling point 56 °C).
  • the volatile liquid needs to act as a solvent for the wall forming material and be miscible with water at the ratios used.
  • preferred capsules may comprise albumin.
  • the proportion of the aqueous solution which is the volatile liquid will typically vary according to the identity of the volatile compound, the concentration and identity of the wall-forming material, the temperature and pressures at which the solution is to be sprayed, and the microcapsule product desired. Typically, between 0.1 % and 80% v/v, preferably 1- 50% v/v and most preferably 5-30% v/v, for example about 20% v/v, of the solution is the volatile liquid. Mixtures of volatile liquids may be used, in which case these percentages refer to the total content of volatile liquid.
  • the hollow microcapsules may be spray dried from solution, in which case the spray-drying may be a one step process such as to provide the desired microcapsule product immediately.
  • the immediate product may be subjected to further process steps, for example heating to further cross-link and insolubilise the protein shell of the microcapsules. This constitutes a two step process.
  • the total process is carried out under sterile conditions.
  • a protein solution is sterile and non-pyrogenic
  • the gas in the chamber is first passed through a 0.2 ⁇ m filter
  • the spray-drier is initially autoclaved, and so on.
  • the final product may be sterilised, for example by exposure to ionising radiation.
  • the wall-forming material of microcapsules used in the method of the invention is a water-soluble material, preferably a protein (the term being used to include non-naturally occurring polypeptides and polyamino acids).
  • it may be collagen, gelatin or (serum) albumin, in each case (if the microcapsules are to be administered to humans) preferably of human origin (ie derived from humans or corresponding in structure to the human protein) or polylysine or polyglutamate. It may be human serum albumin (HA) derived from blood donations or from the fermentation of microorganisms (including cell lines) which have been transformed or transfected to express HA.
  • human serum albumin HA
  • simple or complex carbohydrates, simple amino acids or fatty acids can be used, for example lysine, mannitol, dextran, palmitic acid or behenic acid.
  • HA which term includes analogues and fragments of human albumin, for example those of EP-A-322094, and polymers of monomeric albumin
  • EP-A- 201239 and EP-A-286424 All references are included herein by reference.
  • HA "Analogues and fragments" of HA include all polypeptides (i) which are capable of forming a microcapsule in the process of the invention and (ii) of which a continuous region of at least 50% (preferably at least 75 % , 80% , 90% or 95 %) of the amino acid sequence is at least 80% homologous (preferably at least 90% , 95 % or 99% homologous) with a continuous region of at least 50% (preferably 75 % , 80% , 90% or 95 %) of a nature-identical human albumin.
  • HA which is produced by recombinant DNA techniques may be used.
  • the HA may be produced by expressing an HA-encoding nucleotide sequence in yeast or in another microorganism and purifying the product, as is known in the art.
  • Such material lacks the fatty acids associated with serum-derived material.
  • the HA is substantially free of fatty acids; ie it contains less than 1 % of the fatty acid level of serum-derived material.
  • fatty acid is undetectable in the HA.
  • the aqueous solution or dispersion of microcapsules used according to the invention is preferably 0.1 to 50% w/v, more preferably about 1.0 - 25.0% w/v or 5.0 - 30.0% w/v protein, particularly when the material is albumin. About 5-15 % w/v is optimal. Mixtures of wall-forming materials may be used, in which case the percentages in the last two sentences refer to the total content of wall-forming material.
  • the preparation to be sprayed may contain substances other than the wall- forming material, water and volatile liquid.
  • the aqueous phase may contain 1-20% by weight of water-soluble hydrophilic compounds like sugars and polymers as stabilizers, eg poly vinyl alcohol (PVA), poly vinyl pyrrolidone (PVP), polyethylene glycol (PEG), gelatin, polyglutamic acid and polysaccharides such as starch, dextran, agar, xanthan and the like.
  • PVA poly vinyl alcohol
  • PVP poly vinyl pyrrolidone
  • PEG polyethylene glycol
  • gelatin polyglutamic acid
  • polysaccharides such as starch, dextran, agar, xanthan and the like.
  • Functional agents may be included in suitable microcapsules, for example at 1.0-40.0% w/w, such as X-ray contrast agents (for example Hexabrix (ioxaglic acid), Optiray (ioversol), Omnipaque (iohexol) or Isovice (iopamidol)) or magnetic resonance imaging agents (for example colloidal iron oxide or gadolinium chelates, eg gadopentetic acid).
  • X-ray contrast agents for example Hexabrix (ioxaglic acid), Optiray (ioversol), Omnipaque (iohexol) or Isovice (iopamidol)
  • magnetic resonance imaging agents for example colloidal iron oxide or gadolinium chelates, eg gadopentetic acid.
  • Similar aqueous phases can be used as the carrier liquid in which the final microcapsule product for use according to the invention is suspended before use.
  • Surfactants may be used (0.1- 5 % by weight) including most physiologically acceptable surfactants, for instance egg lecithin or soya bean lecithin, or synthetic lecithins such as saturated synthetic lecithins, for example, dimyristoyl phosphatidyl choline, dipalmitoyl phosphatidyl choline or distearoyl phosphatidyl choline or unsaturated synthetic lecithins, such as dioleyl phosphatidyl choline or dilinoleyl phosphatidyl choline.
  • surfactants include free fatty acids, esters of fatty acids with polyoxyalkylene compounds like polyoxypropylene glycol and polyoxyethylene glycol; ethers of fatty alcohols with polyoxyalkylene glycols; esters of fatty acids with polyoxyalkylated sorbitan; soaps; glycerol-polyalkylene stearate; glycerol-polyoxyethylene ricinoleate; homo- and copolymers of polyalkylene glycols; polyethoxylated soya-oil and castor oil as well as hydrogenated derivatives; ethers and esters of sucrose or other carbohydrates with fatty acids, fatty alcohols, these being optionally polyoxyalkylated; mono-, di- and triglycerides of saturated or unsaturated fatty acids, glycerides or soya-oil and sucrose.
  • the carrier liquid does not contain a surfactant.
  • Additives can be incorporated into the wall of the microcapsules for use according to the invention to modify the physical properties such as dispersibility, elasticity and water permeability.
  • additives which can "hydrophobize" the wall in order to decrease water permeability such as fats, waxes and high molecular- weight hydrocarbons.
  • Additives which increase dispersibility of the microcapsules in the injectable liquid-carrier are amphipathic compounds like the phospholipids; they also increase water permeability and rate of biodegradability.
  • the microcapsules do not contain additives which increase the dispersibility of the microcapsules, as we have found that they are unnecessary, at least when the microcapsules are made of albumin.
  • the quantity of additives to be incorporated in the wall is extremely variable and depends on the needs. In some cases no additive is used at all; in other cases amounts of additives which may reach about 40.0% by weight of the wall are possible.
  • microcapsules means hollow particles enclosing a space, which space is filled with a gas or vapor but not with any solid materials. Honeycombed particles resembling the confectionery sold in the UK as “Maltesers” (trademark) are not formed. It is not necessary for the space to be totally enclosed (although this is preferred) and it is not necessary for the microcapsules to be precisely spherical, although they are generally spherical.
  • the diameters referred to above relate to the diameter of a corresponding spherical microcapsule having the same mass and enclosing the same volume of hollow space as the non-spherical microcapsule.
  • the final product measured in the same way as the intermediate microcapsules, may, if one wishes, consist of microcapsules having a diameter of 0.1 to 50.0 ⁇ m, but volume ranges of 0.1 to 20.0 ⁇ m and especially 1.0 to 8.0 ⁇ m are obtainable, and are preferred for the method of the invention.
  • the second step may alter the size of the microcapsules in determining the size produced in the first step.
  • a product having a high degree of reflectivity, relative to the amount of wall-forming material is preferred for the method of the invention.
  • a homogeneous suspension of 13 ⁇ g/ml of microcapsules can provide a reflectivity to 3.5MHz ultrasound of at least -1.0 dB. Higher reflectivities than -0.3 may be unnecessary, and a reflectivity of around -0.7 to -0.5 is convenient.
  • microcapsules for use in the method of the present invention can be stored dry in the presence or in the absence of additives to improve conservation, prevent coalescence or aid resuspension.
  • additives one may select from 0.1 to 200.0% by weight of water-soluble physiologically acceptable compounds such as mannitol, galactose, lactose or sucrose or hydrophilic polymers like dextran, xanthan, agar, starch, PVP, polyglutamic acid, polyvinylalcohol (PVA) and gelatin.
  • the useful life- time of the microcapsules in the mjectable liquid carrier phase ie the period during which useful echographic signals are observed, can be controlled to last from a few minutes to several months depending on the needs; this can be done by controlling the porosity, solubility or degree of cross-linking of the wall. These parameters can be controlled by properly selecting the wall-forming materials and additives and by adjusting the evaporation rate and temperature in the spray-drying chamber.
  • the microcapsules can be milled with a suitable inert excipient using a Fritsch centrifugal pin mill equipped with a 0.5 mm screen, or a Glen Creston air impact jet mill.
  • Suitable excipients are finely milled powders which are inert and suitable for intravenous use, such as lactose, glucose, mannitol, sorbitol, galactose, maltose or sodium chloride.
  • the microcapsules/excipient mixture can be suspended in aqueous medium to facilitate removal of non-functional/defective microcapsules, or it can be placed in final containers for distribution without further processing.
  • a trace amount of surfactant can be included in the milling stage and/or in the aqueous medium to prevent agglomeration.
  • Anionic, cationic and non-ionic surfactants suitable for this purpose include poloxamers, sorbitan esters, polysorbates and lecithin.
  • microcapsule suspension may then be allowed to float, or may be centrifuged to sediment any defective particles which have surface defects which would, in use, cause them to fill with liquid and be no longer echogenic.
  • microcapsule suspension may then be remixed to ensure even particle distribution, washed and reconstituted in a buffer suitable for intravenous injection such as isotonic mannitol.
  • a buffer suitable for intravenous injection such as isotonic mannitol.
  • the suspension may be aliquoted for freeze drying and subsequent sterilisation by, for example, gamma irradiation, dry heating or ethylene oxide.
  • An alternative method for deagglomeration of the insolubilised or fixed microcapsules is to suspend them directly in an aqueous medium containing a suitable surfactant, for example poloxamers, sorbitan esters, polysorbates and lecithin. Deagglomeration may then be achieved using a suitable homogeniser. The microcapsule suspension may then be allowed to float or may be centrifuged to sediment the defective particles, as above, and further treated as above.
  • a suitable surfactant for example poloxamers, sorbitan esters, polysorbates and lecithin.
  • the product of the heat fixing step is de-agglomerated by milling as above.
  • compositions for use in the method according to the invention may conveniently be intravenously administered, either by continual drip feed or as a bolus.
  • the techniques for use in performing the method of the invention may utilize ultrasonic scanning equipment, which may consist of a scanner and imaging apparatus. Such equipment is typically used to produce visual images of a predetermined area, such as the heart region of a human body. Typically, a transducer is placed directly on the skin over the area to be imaged. A scanner houses various electronic components including ultrasonic transducers. The transducer produces ultrasonic waves which perform a sector scan of the heart region. The ultrasonic waves are reflected by the various portions of the heart region and are received by the receiving transducer and processed in accordance with the pulse-echo methods known in the art. After processing, signals are sent to the imaging apparatus (also well known in the art) for viewing. As discussed above, the method of the invention utilizes electrocardiograph measurements for detecting ventricular premature beats.
  • the microcapsule suspension is injected, for example through an arm vein.
  • the microcapsules flow through the vein into the right venous side of the heart, through the main pulmonary artery leading to the lungs, across the lungs, through the capillaries, into the pulmonary vein and finally into the left atrium and the left ventricular cavity of the heart.
  • the hollow microcapsules of the invention induce in the patient a ventricular premature beat which may be anything up to 20 beats/minute.
  • DCE dynamic cross-sectional echography
  • Types of apparatus utilised in DCE are generally called DCE scanners and transmit and receive short, sonic pulses in the form of narrow beams or lines.
  • the reflected signals' strength is a function of time, which is converted to a position using a nominal sound speed, and is displayed on a cathode ray tube or other suitable devices in a manner somewhat analogous to radar or sonar displays.
  • Hollow "Quantison” microcapsules (Andaris, Nottingham, UK), made by the method of W096/15814, are administered intravenously to a patient by both infusion and bolus injection.
  • Ultrasound was administered 5 minutes after the start of the infusion and maintained for 15 minutes, and also for a period of 5-10 minutes following each bolus injection.
  • Ultrasound treatment is carried out at a Mechanical Index between 0.5 - 5.0, preferably as low as possible in the range 1.5-3.0 MHz, and ideally 1.5.
  • the induction of ultrasound is by triggered emission.
  • the ultrasound beam is passed through the heart in a plane at any angle, and premature beats induced; ventricular premature beats are detected by electrocardiography .
  • the harmonic mode may be utilized, and the compression should be set at the minimum.
  • the dynamic range can be set at 60dB (ATL300), and an apical 4 chamber view can be used.
  • the focal depth of the machine should conveniently be set slightly beyond the region of interest, ie in line with the mitral valve, and the gain settings should conveniently be set such that the myocardium appears uniformly echoreflective across the whole cross-section with a relatively higher grey scale than is usual in normal echocardiographic studies.
  • Induced ventricular premature beats were found to occur only during end- systolic triggering, with an individual sensitivity to the occurrence of ventricular premature beats. In a comparison between infusion and bolus injection, the chances of inducing ventricular premature beats were found to be higher during infusion. Furthermore, the amount of acoustic energy is related to induction of ventricular premature beats, with higher acoustic pressure inducing more premature beats. Switching of the ultrasound beam resulted in acute cessation of induced ventricular premature beats.
  • the ultrasound contrast agent AIPIOI (Andaris Ltd, Nottingham, United Kingdom) was used in both studies. It consists of heat stabilized, air-filled albumin microcapsules with a median diameter of 4 ⁇ m.
  • AIPIOI was formulated with glucose as excipient containing 1500 x 10 6 microcapsules/ml after reconstitution with 5ml water for injection.
  • mannitol was formulated with excipient, containing 500 x 10 6 microcapsules/ml after reconstitution.
  • Imaging started with obtaining a baseline apical 4 chamber, 2 chamber, and 3 chamber view in triggered second harmonic mode with a mechanical index of 1.5. Triggering was done end-systolic (at the end of T-wave), one scan per heart cycle. Imaging in apical 4 chamber view continued through infusion until 5 minutes after the end of infusion. At the end of infusion, end-systolic and end-diastolic triggered images of apical 2 chamber and 3 chamber view, and parasternal long axis and parasternal short axis were obtained for 5 to 15 seconds each. In volunteers 7 to 10 infusion was started with glucose 5 % for five minutes prior to contrast infusion, while imaging continued. All images were stored on S-VHS tape, together with one channel monitor ECG.
  • Infusion was done with an infusion rate of lml/min for 20 minutes in subjects 1 to 4 and for 10 minutes in subjects 5 to 10.
  • AIP 101 was diluted with 5 % glucose to obtain microcapsule infusion rates between 25 and 750 x 10° microcapsules/min.
  • Imaging started with obtaining baseline apical 4 chamber view images in triggered second harmonic mode with a mechanical index of 1.1 and 1.5. Triggering was done end-systole (at the end of the T-wave) or end-diastole (at the first deflection of the QRS-complex), one scan per heart cycle.
  • the infusion protocol continued with 5 minutes of end-systolic triggered imaging with an Ml of 1.5 without contrast infusion, followed by 5 minutes of contrast infusion with an infusion rate of lml/min without imaging.
  • bolus injections were given as a 1 ml rapid intravenous injection, followed by a 5 ml saline flush over 3 seconds.
  • the time interval between bolus injections was at least 5 minutes.
  • one of three imaging modalities was used: end-systolic triggering and an MI of 1.1 or 1.5, or end-diastolic with an MI of 1.5, while the sequence of imaging modes was varied between subjects.
  • the time interval between QRS complex and trigger moment for end-systolic triggering varied between 350 and 420 msec between individual studies, but was kept constant during each study. All images were stored on S-VHS tape, together with one channel monitor ECG.
  • the efficacy of the contrast agent for myocardial opacification was visually assessed and scored for 6 segments in the apical 4 chamber view. Segments were scored as not visible, just visible, and good opacification by two experienced operators on selected digitized images side-by side with digitized baseline images.
  • a continuous single lead ECG was stored together with the ultrasound images on S-VHS video tape.
  • a continuous 6 or 12 lead ECG was obtained throughout the bolus versus infusion study. Blood pressure was recorded every 5 minutes throughout the study, and blood samples for blood chemistry and hematology were obtained before the study and 1 hour after the last contrast administration.
  • the maximum PVC rate during any given minute was 16/min, while the most severe arrhythmia was a run of 5 multiform PVC's with a frequency of 130 bpm.
  • the occurrence of PVC's was slightly uncomfortable, subsided after cessation of imaging, and was considered a minor (non-serious) side effect in these volunteers.
  • the majority (98 %) of PVC's occurred with a coupling interval between the preceding triggered image and the first deflection of the PVC of 80 ⁇ 10 msec. Origin of PVC
  • PVC's Two hundred and ten PVC's were recorded on six or twelve lead EKG in the bolus versus infusion study. Fifty five (26%) had a left bundle branch block configuration, while 154 (73 %) had a right bundle branch block configuration. The electrical axis was distributed over all four quadrants, with right axis deviation the most common configuration (44%). PVC's were not uniform, neither within volunteers, nor between volunteers. No single region could be established from which the PVC's originated. Although total imaging time in other than the apical four chamber view was limited, the PVC rate in parasternal views was higher compared to apical views in the dose-ranging study (1.8 vs 7.1/min). However, no 12- lead EG was available in this study, allowing no inference about right ventricular involvement in the origin of these PVC's.
  • the mean PVC rate with bolus injection was 0.06/min, while it was 0.60/min with continuous infusion.
  • a significant increase in PVC was observed during end-systolic triggered imaging with an MI of 1.5 in the presence of the contrast agent (1.6 PVC/min, p ⁇ 0.001).
  • Most PVC's (92%) were observed during end-systolic triggering with an MI of 1.5, with a higher rate during infusion of contrast (2.2/min) as compared with bolus injection (0.5/min).

Abstract

Hollow microcapsules, preferably made by spray-drying a protein, are used to induce ventricular premature beats in the heart, as an aid to diagnosing heart conditions.

Description

METHOD OF ALTERING HEARTBEAT
The present invention relates to a method of inducing a ventricular premature heart beat in a human or animal patient. Such induction may be beneficial in the medical field, in relation to the diagnosis of coronary arrhythmias. The method utilizes the introduction into the patient of hollow microcapsules, and their interaction with ultrasound.
Air bubbles are known in the art for use in echocardiography techniques, in which a combination of hollow microcapsules intravenously injected into the body and ultrasound techniques are used to investigate the heart. WO 92/18164 discloses the spray drying of a solution of a wall forming material, preferably a protein such as albumin, to form microcapsules. Further, in WO 96/15814, there is disclosed a method for forming microcapsules, which can be used as an echogenic contrast agent, which involves providing a solution of a material (which is advantageously a protein) in a solvent which comprises water and a liquid of greater volatility than water, and spraying the solution into a gas to evaporate the aqueous solvent, thereby providing hollow microcapsules.
In utilising microcapsules of the type described in WO 96/15814 in echocardiography techniques, we have surprisingly found that the administering of such microcapsules can induce in patients a ventricular premature beat. The monitoring and observation of such a ventricular premature beat can provide useful information on the patient, and in particular can be used to investigate ventricular tachycardia, and identify the source of an ectopic beat. Such a method represents a highly advantageous advance on other diagnostic methods for ventricular tachycardia, which currently induce ventricular premature beats either by invasive techniques including catheter placement, or by gross electrical stimulation to the thorax. Further, the stimulation of ventricular premature beats in patients can be used to identify arrhythmias, and the type and origin of arrhythmia involved. They can also be used to determine whether anti-arrhythmic medication administered to patients who have previously experienced arrhythmias, ventricular tachycardia or ventricular fibrillation has been successful.
The method of the invention may also provide a benefit in the testing of automatic implantable cardioverter/defibrillator devices (AICD). The method has the advantage that, unlike invasive methods which cannot be used too often, it can in fact be repeated indefinitely.
Ventricular premature beats are also known as premature ventricular contractions or ventricular extrasystoles, and are heartbeats where the electrical origin or pacemaker is within the ventricular myocardium, as opposed to a sinus beat or supraventricular beat, where the electrical origin lies within the atrial myocardium. Such a beat also falls prior to when the next beat in a regular heart rhythm is expected, in contrast to an escape beat.
Further, ventricular premature beats appear prematurely during regular sinus rhythm or any other non-ventricular rhythm, and are characterised by broad QRS-complexes ( >0.11s) without a preceding p-wave, or more precisely, without a preceding His bundel signal, which cannot normally be observed on a 12-channel electrocardiograph. Such beats occur in normal healthy hearts, as well as under pathological conditions. In the latter case, they may precede or induce ventricular tachycardia. In particular in ischemic hearts, this may lead to life threatening situations.
Thus, according to a first aspect of the invention, there is provided a method of inducing in a human or animal patient a ventricular premature beat, comprising intravenously administering thereto a composition containing hollow microcapsules, and exposing the patient to ultrasound.
According to a further aspect of the invention there is provided the use of hollow microcapsules for the manufacture of a diagnostic agent for the induction of ventricular premature beats in a patient.
According to yet a further aspect of the invention, there is provided a method of inducing and detecting in a human or animal patient a ventricular premature beat, comprising intravenously administering thereto a hollow microcapsule, exposing the patient to ultrasound, and examining the patient to detect the presence or otherwise of a ventricular premature beat.
Conveniently, ventricular premature beats can be observed in the patient using electrocardiograph techniques, in particular conventional 12-channel EKG or body-surface EKG are preferred to detect the presence of the ventricular premature beat, or the induction of ventricular tachycardia.
The method of the invention utilizes ultrasound which conveniently has a Mechanical Index of 1.5, or above, conveniently 1.5, and also systolic triggering in order to significantly increase the number of ventricular premature beats in patients. In the invention, Mechanical Index is the measure of the ultrasound output of a suitable ultrasound device, and is defined as the maximal acoustic pressure in MPa, normally at the focal point, divided by the square root of the ultrasound frequency in MHz.
The technique has been shown to work from both apical and parasternal views.
The method according to the invention may preferably have utility in a substantial proportion of the population - ie it will induce ventricular premature beats in at least 0.5 % , preferably at least 0.7% , more preferably at least 1 % of the population. The method according to the invention may also conveniently be used as a step in a diagnostic method for the types of condition described above.
The method of the invention may be used to induce ventricular premature beats at specific QRS to ventricular premature beat intervals. The QRS to ventricular premature beat interval can be changed by changing the QRS to trigger interval; a ventricular premature beat will occur approximately 80ms after the trigger.
A preferred microcapsule for use in the method of the invention is that described in WO 96/15814. The preferred microcapsules thus are proteinaceous, have a relatively narrow size distribution and thinner shells, and can be made by spray drying an aqueous solution of protein, with the solvent comprising a solvent more volatile than water. Where such microcapsules are used in the method, and the solvent does include a solvent of higher volatility than water, suitable volatile liquids include ethanol (a preferred volatile liquid; boiling point 78.3°C), methanol (boiling point 64.5°C) and acetone (boiling point 56 °C). The volatile liquid needs to act as a solvent for the wall forming material and be miscible with water at the ratios used. As such, preferred capsules may comprise albumin.
The proportion of the aqueous solution which is the volatile liquid will typically vary according to the identity of the volatile compound, the concentration and identity of the wall-forming material, the temperature and pressures at which the solution is to be sprayed, and the microcapsule product desired. Typically, between 0.1 % and 80% v/v, preferably 1- 50% v/v and most preferably 5-30% v/v, for example about 20% v/v, of the solution is the volatile liquid. Mixtures of volatile liquids may be used, in which case these percentages refer to the total content of volatile liquid.
Conveniently the hollow microcapsules may be spray dried from solution, in which case the spray-drying may be a one step process such as to provide the desired microcapsule product immediately. Alternatively, the immediate product may be subjected to further process steps, for example heating to further cross-link and insolubilise the protein shell of the microcapsules. This constitutes a two step process.
Preferably, the total process is carried out under sterile conditions. Thus, a protein solution is sterile and non-pyrogenic, the gas in the chamber is first passed through a 0.2 μm filter, the spray-drier is initially autoclaved, and so on. Alternatively, or as well, the final product may be sterilised, for example by exposure to ionising radiation. The wall-forming material of microcapsules used in the method of the invention is a water-soluble material, preferably a protein (the term being used to include non-naturally occurring polypeptides and polyamino acids). For example, it may be collagen, gelatin or (serum) albumin, in each case (if the microcapsules are to be administered to humans) preferably of human origin (ie derived from humans or corresponding in structure to the human protein) or polylysine or polyglutamate. It may be human serum albumin (HA) derived from blood donations or from the fermentation of microorganisms (including cell lines) which have been transformed or transfected to express HA. Alternatively, simple or complex carbohydrates, simple amino acids or fatty acids can be used, for example lysine, mannitol, dextran, palmitic acid or behenic acid.
Techniques for expressing HA (which term includes analogues and fragments of human albumin, for example those of EP-A-322094, and polymers of monomeric albumin) are disclosed in, for example, EP-A- 201239 and EP-A-286424. All references are included herein by reference. "Analogues and fragments" of HA include all polypeptides (i) which are capable of forming a microcapsule in the process of the invention and (ii) of which a continuous region of at least 50% (preferably at least 75 % , 80% , 90% or 95 %) of the amino acid sequence is at least 80% homologous (preferably at least 90% , 95 % or 99% homologous) with a continuous region of at least 50% (preferably 75 % , 80% , 90% or 95 %) of a nature-identical human albumin. HA which is produced by recombinant DNA techniques may be used. Thus, the HA may be produced by expressing an HA-encoding nucleotide sequence in yeast or in another microorganism and purifying the product, as is known in the art. Such material lacks the fatty acids associated with serum-derived material. Preferably, the HA is substantially free of fatty acids; ie it contains less than 1 % of the fatty acid level of serum-derived material. Preferably, fatty acid is undetectable in the HA.
The aqueous solution or dispersion of microcapsules used according to the invention is preferably 0.1 to 50% w/v, more preferably about 1.0 - 25.0% w/v or 5.0 - 30.0% w/v protein, particularly when the material is albumin. About 5-15 % w/v is optimal. Mixtures of wall-forming materials may be used, in which case the percentages in the last two sentences refer to the total content of wall-forming material.
In a preferred embodiment of microcapsule for use in the invention, the preparation to be sprayed may contain substances other than the wall- forming material, water and volatile liquid. Thus, the aqueous phase may contain 1-20% by weight of water-soluble hydrophilic compounds like sugars and polymers as stabilizers, eg poly vinyl alcohol (PVA), poly vinyl pyrrolidone (PVP), polyethylene glycol (PEG), gelatin, polyglutamic acid and polysaccharides such as starch, dextran, agar, xanthan and the like.
Functional agents may be included in suitable microcapsules, for example at 1.0-40.0% w/w, such as X-ray contrast agents (for example Hexabrix (ioxaglic acid), Optiray (ioversol), Omnipaque (iohexol) or Isovice (iopamidol)) or magnetic resonance imaging agents (for example colloidal iron oxide or gadolinium chelates, eg gadopentetic acid).
Similar aqueous phases can be used as the carrier liquid in which the final microcapsule product for use according to the invention is suspended before use. Surfactants may be used (0.1- 5 % by weight) including most physiologically acceptable surfactants, for instance egg lecithin or soya bean lecithin, or synthetic lecithins such as saturated synthetic lecithins, for example, dimyristoyl phosphatidyl choline, dipalmitoyl phosphatidyl choline or distearoyl phosphatidyl choline or unsaturated synthetic lecithins, such as dioleyl phosphatidyl choline or dilinoleyl phosphatidyl choline. Other surfactants include free fatty acids, esters of fatty acids with polyoxyalkylene compounds like polyoxypropylene glycol and polyoxyethylene glycol; ethers of fatty alcohols with polyoxyalkylene glycols; esters of fatty acids with polyoxyalkylated sorbitan; soaps; glycerol-polyalkylene stearate; glycerol-polyoxyethylene ricinoleate; homo- and copolymers of polyalkylene glycols; polyethoxylated soya-oil and castor oil as well as hydrogenated derivatives; ethers and esters of sucrose or other carbohydrates with fatty acids, fatty alcohols, these being optionally polyoxyalkylated; mono-, di- and triglycerides of saturated or unsaturated fatty acids, glycerides or soya-oil and sucrose. Preferably, however, the carrier liquid does not contain a surfactant.
Additives can be incorporated into the wall of the microcapsules for use according to the invention to modify the physical properties such as dispersibility, elasticity and water permeability.
Among the useful additives, one may cite compounds which can "hydrophobize" the wall in order to decrease water permeability, such as fats, waxes and high molecular- weight hydrocarbons. Additives which increase dispersibility of the microcapsules in the injectable liquid-carrier are amphipathic compounds like the phospholipids; they also increase water permeability and rate of biodegradability. Preferably, however, the microcapsules do not contain additives which increase the dispersibility of the microcapsules, as we have found that they are unnecessary, at least when the microcapsules are made of albumin. The quantity of additives to be incorporated in the wall is extremely variable and depends on the needs. In some cases no additive is used at all; in other cases amounts of additives which may reach about 40.0% by weight of the wall are possible.
The solution of the wall-forming material is atomised and may be spray- dried by any suitable technique which results in discrete microcapsules of 0.05 - 50.0 μm diameter. These figures refer to at least 90% of the volume of microcapsules, the diameter being measured with a Coulter Multisizer II. The term "microcapsules" means hollow particles enclosing a space, which space is filled with a gas or vapor but not with any solid materials. Honeycombed particles resembling the confectionery sold in the UK as "Maltesers" (trademark) are not formed. It is not necessary for the space to be totally enclosed (although this is preferred) and it is not necessary for the microcapsules to be precisely spherical, although they are generally spherical. If the microcapsules are not spherical, then the diameters referred to above relate to the diameter of a corresponding spherical microcapsule having the same mass and enclosing the same volume of hollow space as the non-spherical microcapsule.
Similar provisions apply as in WO 96/15814 with regard to the preparation and atomization of hollow microcapsules and processing conditions, in both the one step and two step procedures, in order to obtain hollow microcapsules having tight size distributions for use according to the invention.
The final product, measured in the same way as the intermediate microcapsules, may, if one wishes, consist of microcapsules having a diameter of 0.1 to 50.0 μm, but volume ranges of 0.1 to 20.0 μm and especially 1.0 to 8.0 μm are obtainable, and are preferred for the method of the invention. One needs to take into account the fact that the second step may alter the size of the microcapsules in determining the size produced in the first step.
In particular, a product having a high degree of reflectivity, relative to the amount of wall-forming material, is preferred for the method of the invention. For example, a homogeneous suspension of 13 μg/ml of microcapsules can provide a reflectivity to 3.5MHz ultrasound of at least -1.0 dB. Higher reflectivities than -0.3 may be unnecessary, and a reflectivity of around -0.7 to -0.5 is convenient.
The microcapsules for use in the method of the present invention can be stored dry in the presence or in the absence of additives to improve conservation, prevent coalescence or aid resuspension. As additives, one may select from 0.1 to 200.0% by weight of water-soluble physiologically acceptable compounds such as mannitol, galactose, lactose or sucrose or hydrophilic polymers like dextran, xanthan, agar, starch, PVP, polyglutamic acid, polyvinylalcohol (PVA) and gelatin. The useful life- time of the microcapsules in the mjectable liquid carrier phase, ie the period during which useful echographic signals are observed, can be controlled to last from a few minutes to several months depending on the needs; this can be done by controlling the porosity, solubility or degree of cross-linking of the wall. These parameters can be controlled by properly selecting the wall-forming materials and additives and by adjusting the evaporation rate and temperature in the spray-drying chamber.
In order to minimize any agglomeration of the microcapsules, the microcapsules can be milled with a suitable inert excipient using a Fritsch centrifugal pin mill equipped with a 0.5 mm screen, or a Glen Creston air impact jet mill. Suitable excipients are finely milled powders which are inert and suitable for intravenous use, such as lactose, glucose, mannitol, sorbitol, galactose, maltose or sodium chloride. Once milled, the microcapsules/excipient mixture can be suspended in aqueous medium to facilitate removal of non-functional/defective microcapsules, or it can be placed in final containers for distribution without further processing. To facilitate subsequent reconstitution in the aqueous phase, a trace amount of surfactant can be included in the milling stage and/or in the aqueous medium to prevent agglomeration. Anionic, cationic and non-ionic surfactants suitable for this purpose include poloxamers, sorbitan esters, polysorbates and lecithin.
The microcapsule suspension may then be allowed to float, or may be centrifuged to sediment any defective particles which have surface defects which would, in use, cause them to fill with liquid and be no longer echogenic.
The microcapsule suspension may then be remixed to ensure even particle distribution, washed and reconstituted in a buffer suitable for intravenous injection such as isotonic mannitol. The suspension may be aliquoted for freeze drying and subsequent sterilisation by, for example, gamma irradiation, dry heating or ethylene oxide.
An alternative method for deagglomeration of the insolubilised or fixed microcapsules is to suspend them directly in an aqueous medium containing a suitable surfactant, for example poloxamers, sorbitan esters, polysorbates and lecithin. Deagglomeration may then be achieved using a suitable homogeniser. The microcapsule suspension may then be allowed to float or may be centrifuged to sediment the defective particles, as above, and further treated as above.
In a preferred embodiment of the invention, the product of the heat fixing step is de-agglomerated by milling as above.
The compositions for use in the method according to the invention may conveniently be intravenously administered, either by continual drip feed or as a bolus.
The techniques for use in performing the method of the invention may utilize ultrasonic scanning equipment, which may consist of a scanner and imaging apparatus. Such equipment is typically used to produce visual images of a predetermined area, such as the heart region of a human body. Typically, a transducer is placed directly on the skin over the area to be imaged. A scanner houses various electronic components including ultrasonic transducers. The transducer produces ultrasonic waves which perform a sector scan of the heart region. The ultrasonic waves are reflected by the various portions of the heart region and are received by the receiving transducer and processed in accordance with the pulse-echo methods known in the art. After processing, signals are sent to the imaging apparatus (also well known in the art) for viewing. As discussed above, the method of the invention utilizes electrocardiograph measurements for detecting ventricular premature beats.
In the method of the present invention, after the patient is "prepped" and the scanner is in place, the microcapsule suspension is injected, for example through an arm vein. The microcapsules flow through the vein into the right venous side of the heart, through the main pulmonary artery leading to the lungs, across the lungs, through the capillaries, into the pulmonary vein and finally into the left atrium and the left ventricular cavity of the heart.
Once administered, the hollow microcapsules of the invention induce in the patient a ventricular premature beat which may be anything up to 20 beats/minute.
Besides the scanner briefly described above, there exist other ultrasonic scanners, for use in the method of the invention, examples of which are disclosed in US Patents Nos. 4, 134,554 and 4,315,435. Basically, these patents relate to various techniques including dynamic cross-sectional echography (DCE) for producing sequential two-dimensional images of cross-sectional slices of animal or human anatomy by means of ultrasound energy at a frame rate sufficient to enable dynamic visualisation of moving organs. Types of apparatus utilised in DCE are generally called DCE scanners and transmit and receive short, sonic pulses in the form of narrow beams or lines. The reflected signals' strength is a function of time, which is converted to a position using a nominal sound speed, and is displayed on a cathode ray tube or other suitable devices in a manner somewhat analogous to radar or sonar displays.
Preferred aspects of the invention will now be described by way of example only, with reference to the accompanying drawing, in which Figure 1 represents a graph of PVC's versus time in the experiment outlined in Example 2. Example 1 - Protocol for induction of ventricular premature beats
Hollow "Quantison" microcapsules (Andaris, Nottingham, UK), made by the method of W096/15814, are administered intravenously to a patient by both infusion and bolus injection.
Initially 25ml of capsule containing solution was administered at a rate of lml/minute providing a dosage level of 3.5-7.0 x 106 microcapsules/kg/minute. After infusion a gap of two hours was left, and each patient then had administered 3 bolus injections, each of volume lml, providing a capsule dosage level of 3.5-7.0 x 106 microcapsules/kg of patient body weight. The bolus injections were separated by 15 minute intervals, with each bolus injection being followed by a 5ml saline flush.
Ultrasound was administered 5 minutes after the start of the infusion and maintained for 15 minutes, and also for a period of 5-10 minutes following each bolus injection.
Ultrasound treatment is carried out at a Mechanical Index between 0.5 - 5.0, preferably as low as possible in the range 1.5-3.0 MHz, and ideally 1.5. The induction of ultrasound is by triggered emission. The ultrasound beam is passed through the heart in a plane at any angle, and premature beats induced; ventricular premature beats are detected by electrocardiography .
With regard to the machine settings on the ultrasound equipment, the harmonic mode may be utilized, and the compression should be set at the minimum. The dynamic range can be set at 60dB (ATL300), and an apical 4 chamber view can be used. The focal depth of the machine should conveniently be set slightly beyond the region of interest, ie in line with the mitral valve, and the gain settings should conveniently be set such that the myocardium appears uniformly echoreflective across the whole cross-section with a relatively higher grey scale than is usual in normal echocardiographic studies.
In a study to induce ventricular premature beats involving nine healthy subjects treated in accordance with the above protocol, all nine subjects exhibited ventricular premature beats during infusion and imaging. All of the subjects exhibited less than 20 ventricular premature beats per minute. The number of extra systole ranged from 1 to 14 per minute, with a mean of 2.4 per minute.
Induced ventricular premature beats were found to occur only during end- systolic triggering, with an individual sensitivity to the occurrence of ventricular premature beats. In a comparison between infusion and bolus injection, the chances of inducing ventricular premature beats were found to be higher during infusion. Furthermore, the amount of acoustic energy is related to induction of ventricular premature beats, with higher acoustic pressure inducing more premature beats. Switching of the ultrasound beam resulted in acute cessation of induced ventricular premature beats.
Example 2
Subjects
Two open label studies in healthy male volunteers were performed, the first a dose-ranging study with continuous infusion of the contrast agent, the second a comparison between bolus injection and continuous infusion of the contrast agent. In both studies the healthy condition of subjects was established by medical history, physical examination, ECG, blood and urine samples prior to the investigation. Furthermore all subjects were screened for optimal echo window. The protocols were approved by the Medical Ethical Committee of the Academic Medical Center. All subjects gave written informed consent.
Ultrasound contrast agent
The ultrasound contrast agent AIPIOI (Andaris Ltd, Nottingham, United Kingdom) was used in both studies. It consists of heat stabilized, air-filled albumin microcapsules with a median diameter of 4 μm. In the dose- ranging study AIPIOI was formulated with glucose as excipient containing 1500 x 106 microcapsules/ml after reconstitution with 5ml water for injection. In the bolus versus infusion study it was formulated with mannitol as excipient, containing 500 x 106 microcapsules/ml after reconstitution.
Infusion and imaging protocol for dose ranging study
All volunteers received three continuous intravenous infusions with three different doses of AIPIOI , separated by at least 2 hours between end of one infusion and start of the next infusion. Imaging started with obtaining a baseline apical 4 chamber, 2 chamber, and 3 chamber view in triggered second harmonic mode with a mechanical index of 1.5. Triggering was done end-systolic (at the end of T-wave), one scan per heart cycle. Imaging in apical 4 chamber view continued through infusion until 5 minutes after the end of infusion. At the end of infusion, end-systolic and end-diastolic triggered images of apical 2 chamber and 3 chamber view, and parasternal long axis and parasternal short axis were obtained for 5 to 15 seconds each. In volunteers 7 to 10 infusion was started with glucose 5 % for five minutes prior to contrast infusion, while imaging continued. All images were stored on S-VHS tape, together with one channel monitor ECG.
Infusion was done with an infusion rate of lml/min for 20 minutes in subjects 1 to 4 and for 10 minutes in subjects 5 to 10. AIP 101 was diluted with 5 % glucose to obtain microcapsule infusion rates between 25 and 750 x 10° microcapsules/min.
Infusion and imaging protocol for bolus versus infusion study
All volunteers in this study were to receive one continuous intravenous infusion with a dose of 500 x 106 mc/min of AIP for 25 minutes, and three bolus injections of 500 x 106 mc of AIPIOI . The infusion and the series of bolus injections were separated by at least 2 hours, while a minimum of five minutes between bolus injections was observed.
Imaging started with obtaining baseline apical 4 chamber view images in triggered second harmonic mode with a mechanical index of 1.1 and 1.5. Triggering was done end-systole (at the end of the T-wave) or end-diastole (at the first deflection of the QRS-complex), one scan per heart cycle. The infusion protocol continued with 5 minutes of end-systolic triggered imaging with an Ml of 1.5 without contrast infusion, followed by 5 minutes of contrast infusion with an infusion rate of lml/min without imaging. In the next 20 minutes contrast infusion continued, while imaging was done for 5 minutes each with end-systolic triggering with an MI of 1.5 , end-systolic with an MI of 1.1 , end-diastolic with an MI of 1.1 , and end-diastolic with an MI of 1.5. In 4 volunteers the last sequence as reversed. The infusion was then stopped, and imaging continued for 5 minutes with end-systolic triggering and an MI of 1.5 in all volunteers.
Three bolus injections were given as a 1 ml rapid intravenous injection, followed by a 5 ml saline flush over 3 seconds. The time interval between bolus injections was at least 5 minutes. With each bolus injection one of three imaging modalities was used: end-systolic triggering and an MI of 1.1 or 1.5, or end-diastolic with an MI of 1.5, while the sequence of imaging modes was varied between subjects. The time interval between QRS complex and trigger moment for end-systolic triggering varied between 350 and 420 msec between individual studies, but was kept constant during each study. All images were stored on S-VHS tape, together with one channel monitor ECG.
Evaluation of efficacy
The efficacy of the contrast agent for myocardial opacification was visually assessed and scored for 6 segments in the apical 4 chamber view. Segments were scored as not visible, just visible, and good opacification by two experienced operators on selected digitized images side-by side with digitized baseline images.
Safety monitoring
A continuous single lead ECG was stored together with the ultrasound images on S-VHS video tape. A continuous 6 or 12 lead ECG was obtained throughout the bolus versus infusion study. Blood pressure was recorded every 5 minutes throughout the study, and blood samples for blood chemistry and hematology were obtained before the study and 1 hour after the last contrast administration.
Ultrasound machine
An HDI 3000 (Advanced Technology Laboratories, Bothwell, WA) was used for all imaging. Custom-made software allowed a mechanical index of 1.5 in triggered mode. Contrast specific imaging with second harmonic filtering was used throughout both studies.
Results
A total of 19 healthy male volunteers were recruited: ten volunteers for the dose-ranging study, and 9 for the bolus versus infusion study. The mean age was 27.8 ± 5.7 years (range 19-41). In these 19 volunteers a total of 967 premature ventricular contractions (PVC's) were observed over a total observation period of 29.3 hours (0.55 PVC/min). The total number of PVC's during baseline imaging was 10 over 5.7 hours (0.03 PVC/min), while 944 PVC's (97.6%) occurred during 14.9 hours of systolic triggered contrast imaging with an MI of 1.5 (1.06 PVC/min). Individual PVC rate ranged from 0.0 to 8.0 PVC/min during a study period. The maximum PVC rate during any given minute was 16/min, while the most severe arrhythmia was a run of 5 multiform PVC's with a frequency of 130 bpm. The occurrence of PVC's was slightly uncomfortable, subsided after cessation of imaging, and was considered a minor (non-serious) side effect in these volunteers. The majority (98 %) of PVC's occurred with a coupling interval between the preceding triggered image and the first deflection of the PVC of 80 ± 10 msec. Origin of PVC
Two hundred and ten PVC's were recorded on six or twelve lead EKG in the bolus versus infusion study. Fifty five (26%) had a left bundle branch block configuration, while 154 (73 %) had a right bundle branch block configuration. The electrical axis was distributed over all four quadrants, with right axis deviation the most common configuration (44%). PVC's were not uniform, neither within volunteers, nor between volunteers. No single region could be established from which the PVC's originated. Although total imaging time in other than the apical four chamber view was limited, the PVC rate in parasternal views was higher compared to apical views in the dose-ranging study (1.8 vs 7.1/min). However, no 12- lead EG was available in this study, allowing no inference about right ventricular involvement in the origin of these PVC's.
Effect of mechanical index and trigger moment
During imaging PVC's were almost exclusively observed when triggering end-systolic, and with an MI of 1.5. End-diastolic triggering or end- systolic triggering with an MI of 1.1 did not increase the occurrence of PVC's (0.04 and 0.01 PVC/min).
Effect of infusion dose
In the dose-ranging smdy a relation between infusion rate and occurrence of PVC's was observed, with higher doses associated with more PVC's. For this analysis the infusion rates used in each subject were classified as low, medium and high. The PVC rate observed during baseline and with low, medium and high infusion rates were 0.02, 0.29, 0.80 and 1.83/min respectively (p < 0.001). After an initial increase, the PVC rate decreased again in the last 10 minutes of infusion in those subjects who had 20 minutes of infusion with higher doses, possibly due to attenuation caused by the higher concentration that resulted from prolonged infusion, as is shown in Figure 1.
Bolus versus infusion
The mean PVC rate with bolus injection was 0.06/min, while it was 0.60/min with continuous infusion. A significant increase in PVC was observed during end-systolic triggered imaging with an MI of 1.5 in the presence of the contrast agent (1.6 PVC/min, p < 0.001). Most PVC's (92%) were observed during end-systolic triggering with an MI of 1.5, with a higher rate during infusion of contrast (2.2/min) as compared with bolus injection (0.5/min). Neither ultrasound with a high MI without contrast nor the contrast agent without ultrasound led to increase in PVC rate (0.05 and 0.07 PVC/min respectively).
Efficacy
Contrast was observed in the myocardium in all subjects. 29% of segments were scored as full opacification, 49% of segments as partial opacification and 22% of segments as no opacification. Imaging with an MI of 1.5 yielded slightly better results as compared to imaging with an MI of 1.1 (88 % full and partial opacification versus 78 % full and partial opacification, p =0.08).

Claims

1. A method of inducing in a human or animal patient a ventricular premamre beat, comprising intravenously administering thereto a hollow microcapsule. and exposing the patient to ultrasound.
2. A method according to Claim 1, wherein the hollow microcapsules are formed by spray-drying a solution of material in an aqueous solution which contains a liquid of greater volatility than water.
3. A method according to Claim 2 wherein the material is a protein.
4. A method according to Claim 3 wherein the protein is albumin, collagen or gelatin.
5. A method according to any one of Claims 2 to 4 wherein the microcapsules are further processed before use, for example by heat treatment to further insoluble the microcapsules, or radiation to sterilise the microcapsules.
6. A method according to any one of claims 1 to 5 wherein the ventricular premature beat is measured by electrocardiograph techniques.
7. A method according to claim 6 wherein the electrocardiograph technique involves the use of conventional EKG or body surface EKG.
8. A method of providing an image of a part of a human or animal patient having a ventricular premature beat, comprising (i) introducing hollow microcapsules, (ii) passing ultrasound radiation into the patient and (iii) creating an image based on the reflectivity, transmissibility or resonance of the microcapsules in the said part of the patient.
9. Use in a diagnostic method of a method according to any of claims 1 to 7.
10. Use of hollow microcapsules for the manufacture of a diagnostic agent for the inducing of ventricular premamre beat.
PCT/GB1999/001418 1998-05-23 1999-05-21 Method of altering heartbeat WO1999061058A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU39382/99A AU3938299A (en) 1998-05-23 1999-05-21 Method of altering heartbeat

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GBGB9811116.4A GB9811116D0 (en) 1998-05-23 1998-05-23 Method of altering heartbeat
GB9811116.4 1998-05-23

Related Child Applications (2)

Application Number Title Priority Date Filing Date
US09/762,761 A-371-Of-International US6589278B1 (en) 1997-05-17 1998-05-15 Vascular prosthesis
US10/603,952 Continuation US9445886B2 (en) 1997-05-17 2003-06-25 Vascular prosthesis

Publications (1)

Publication Number Publication Date
WO1999061058A1 true WO1999061058A1 (en) 1999-12-02

Family

ID=10832598

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB1999/001418 WO1999061058A1 (en) 1998-05-23 1999-05-21 Method of altering heartbeat

Country Status (3)

Country Link
AU (1) AU3938299A (en)
GB (1) GB9811116D0 (en)
WO (1) WO1999061058A1 (en)

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001030397A3 (en) * 1999-10-25 2002-07-11 Univ Nebraska Ultrasound inducement of artial and ventricular rhythms
WO2003070323A1 (en) 2002-02-21 2003-08-28 Technion Research & Development Foundation Ltd. Ultrasound cardiac stimulator
US7006864B2 (en) 2003-06-17 2006-02-28 Ebr Systems, Inc. Methods and systems for vibrational treatment of cardiac arrhythmias
US7050849B2 (en) 2003-11-06 2006-05-23 Ebr Systems, Inc. Vibrational therapy device used for resynchronization pacing in a treatment for heart failure
US7184830B2 (en) 2003-08-18 2007-02-27 Ebr Systems, Inc. Methods and systems for treating arrhythmias using a combination of vibrational and electrical energy
US7558631B2 (en) 2004-12-21 2009-07-07 Ebr Systems, Inc. Leadless tissue stimulation systems and methods
US7606621B2 (en) 2004-12-21 2009-10-20 Ebr Systems, Inc. Implantable transducer devices
US7610092B2 (en) 2004-12-21 2009-10-27 Ebr Systems, Inc. Leadless tissue stimulation systems and methods
US7702392B2 (en) 2005-09-12 2010-04-20 Ebr Systems, Inc. Methods and apparatus for determining cardiac stimulation sites using hemodynamic data
US7765001B2 (en) 2005-08-31 2010-07-27 Ebr Systems, Inc. Methods and systems for heart failure prevention and treatments using ultrasound and leadless implantable devices
US7953493B2 (en) 2007-12-27 2011-05-31 Ebr Systems, Inc. Optimizing size of implantable medical devices by isolating the power source
US8718773B2 (en) 2007-05-23 2014-05-06 Ebr Systems, Inc. Optimizing energy transmission in a leadless tissue stimulation system
US9283392B2 (en) 2008-03-25 2016-03-15 Ebr Systems, Inc. Temporary electrode connection for wireless pacing systems
US9731139B2 (en) 2008-07-16 2017-08-15 Ebr Systems, Inc. Local lead to improve energy efficiency in implantable wireless acoustic stimulators

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992018164A1 (en) * 1991-04-10 1992-10-29 Delta Biotechnology Limited Preparation of diagnostic agents
EP0554213A1 (en) * 1992-01-24 1993-08-04 BRACCO International B.V. Long-lasting aqueous dispersions or suspensions of pressure-resistant gas-filled microvesicles and methods for the preparation thereof
WO1996015814A1 (en) * 1994-11-19 1996-05-30 Andaris Limited Preparation of hollow microcapsules
WO1997044067A2 (en) * 1996-05-23 1997-11-27 Andaris Limited Use of hollow microcapsules
WO1997046264A2 (en) * 1996-06-07 1997-12-11 Molecular Biosystems, Inc. Pressure resistant protein microspheres as ultrasonic imaging agents
WO1998032378A1 (en) * 1997-01-22 1998-07-30 Quadrant Healthcare (Uk) Limited Ultrasound contrast imaging
WO1999008714A1 (en) * 1997-08-19 1999-02-25 Marsden, John, Christopher Improvements in or relating to contrast agents

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992018164A1 (en) * 1991-04-10 1992-10-29 Delta Biotechnology Limited Preparation of diagnostic agents
EP0554213A1 (en) * 1992-01-24 1993-08-04 BRACCO International B.V. Long-lasting aqueous dispersions or suspensions of pressure-resistant gas-filled microvesicles and methods for the preparation thereof
WO1996015814A1 (en) * 1994-11-19 1996-05-30 Andaris Limited Preparation of hollow microcapsules
WO1997044067A2 (en) * 1996-05-23 1997-11-27 Andaris Limited Use of hollow microcapsules
WO1997046264A2 (en) * 1996-06-07 1997-12-11 Molecular Biosystems, Inc. Pressure resistant protein microspheres as ultrasonic imaging agents
WO1998032378A1 (en) * 1997-01-22 1998-07-30 Quadrant Healthcare (Uk) Limited Ultrasound contrast imaging
WO1999008714A1 (en) * 1997-08-19 1999-02-25 Marsden, John, Christopher Improvements in or relating to contrast agents

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
COSGROVE D.: "Echo enhancers and ultrasound imaging.", EUROPEAN JOURNAL OF RADIOLOGY, (1997) 26/1 (64-76)., XP002112634 *
DE JONG N ET AL: "New ultrasound contrast agents and technological innovations", ULTRASONICS, vol. 34, no. 2, 1 June 1996 (1996-06-01), pages 587-590, XP004035697, ISSN: 0041-624X *
FRINKING, PETER J. A. (1) ET AL: "Scattering properties of encapsulated gas bubbles at high ultrasound pressures.", JOURNAL OF THE ACOUSTICAL SOCIETY OF AMERICA, (MARCH, 1999) VOL. 105, NO. 3, PP. 1989-1996., XP002112375 *
KAUL S.: "Myocardial contrast echocardiography in acute myocardial infarction: Time to test for routine clinical use? Editorial.", HEART, (1999) 81/1 (2-5)., XP002112635 *

Cited By (31)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6439236B1 (en) 1999-10-25 2002-08-27 The Board Of Regents Of The University Of Nebraska Methods for inducing atrial and ventricular rhythms using ultrasound and microbubbles
WO2001030397A3 (en) * 1999-10-25 2002-07-11 Univ Nebraska Ultrasound inducement of artial and ventricular rhythms
US7699778B2 (en) 2002-02-21 2010-04-20 Technion Research & Development Foundation Ltd. Ultrasound cardiac stimulator
WO2003070323A1 (en) 2002-02-21 2003-08-28 Technion Research & Development Foundation Ltd. Ultrasound cardiac stimulator
US7006864B2 (en) 2003-06-17 2006-02-28 Ebr Systems, Inc. Methods and systems for vibrational treatment of cardiac arrhythmias
US7184830B2 (en) 2003-08-18 2007-02-27 Ebr Systems, Inc. Methods and systems for treating arrhythmias using a combination of vibrational and electrical energy
US7809438B2 (en) 2003-08-18 2010-10-05 Ebr Systems, Inc. Methods and systems for treating arrhythmias using a combination of vibrational and electrical energy
US7050849B2 (en) 2003-11-06 2006-05-23 Ebr Systems, Inc. Vibrational therapy device used for resynchronization pacing in a treatment for heart failure
US9333364B2 (en) 2004-06-15 2016-05-10 Ebr Systems, Inc. Methods and systems for heart failure treatments using ultrasound and leadless implantable devices
US7606621B2 (en) 2004-12-21 2009-10-20 Ebr Systems, Inc. Implantable transducer devices
US7996087B2 (en) 2004-12-21 2011-08-09 Ebr Systems, Inc. Leadless tissue stimulation systems and methods
US7558631B2 (en) 2004-12-21 2009-07-07 Ebr Systems, Inc. Leadless tissue stimulation systems and methods
US7610092B2 (en) 2004-12-21 2009-10-27 Ebr Systems, Inc. Leadless tissue stimulation systems and methods
US7848815B2 (en) 2004-12-21 2010-12-07 Ebr Systems, Inc. Implantable transducer devices
US7890173B2 (en) 2004-12-21 2011-02-15 Ebr Systems, Inc. Implantable transducer devices
US9008776B2 (en) 2004-12-21 2015-04-14 Ebr Systems, Inc. Leadless tissue stimulation systems and methods
US8315701B2 (en) 2004-12-21 2012-11-20 Ebr Systems, Inc. Leadless tissue stimulation systems and methods
US10207115B2 (en) 2005-08-31 2019-02-19 Ebr Systems, Inc. Method and systems for heart failure prevention and treatments using ultrasound and leadless implantable devices
US7765001B2 (en) 2005-08-31 2010-07-27 Ebr Systems, Inc. Methods and systems for heart failure prevention and treatments using ultrasound and leadless implantable devices
US11376439B2 (en) 2005-08-31 2022-07-05 Ebr Systems, Inc. Methods and systems for heart failure prevention and treatments using ultrasound and leadless implantable devices
US7702392B2 (en) 2005-09-12 2010-04-20 Ebr Systems, Inc. Methods and apparatus for determining cardiac stimulation sites using hemodynamic data
US8718773B2 (en) 2007-05-23 2014-05-06 Ebr Systems, Inc. Optimizing energy transmission in a leadless tissue stimulation system
US11452879B2 (en) 2007-05-23 2022-09-27 Ebr Systems, Inc. Optimizing energy transmission in a leadless tissue stimulation system
US10456588B2 (en) 2007-05-23 2019-10-29 Ebr Systems, Inc. Optimizing energy transmission in a leadless tissue stimulation system
US10080903B2 (en) 2007-05-23 2018-09-25 Ebr Systems, Inc. Optimizing energy transmission in a leadless tissue stimulation system
US7953493B2 (en) 2007-12-27 2011-05-31 Ebr Systems, Inc. Optimizing size of implantable medical devices by isolating the power source
US9907968B2 (en) 2008-03-25 2018-03-06 Ebr Systems, Inc. Temporary electrode connection for wireless pacing systems
US10688307B2 (en) 2008-03-25 2020-06-23 Ebr Systems, Inc. Temporary electrode connection for wireless pacing systems
US9283392B2 (en) 2008-03-25 2016-03-15 Ebr Systems, Inc. Temporary electrode connection for wireless pacing systems
US11752352B2 (en) 2008-03-25 2023-09-12 Ebr Systems, Inc. Temporary electrode connection for wireless pacing systems
US9731139B2 (en) 2008-07-16 2017-08-15 Ebr Systems, Inc. Local lead to improve energy efficiency in implantable wireless acoustic stimulators

Also Published As

Publication number Publication date
GB9811116D0 (en) 1998-07-22
AU3938299A (en) 1999-12-13

Similar Documents

Publication Publication Date Title
Shapiro et al. Intravenous contrast echocardiography with use of sonicated albumin in humans: systolic disappearance of left ventricular contrast after transpulmonary transmission
US5701899A (en) Perfluorobutane ultrasound contrast agent and methods for its manufacture and use
US5695740A (en) Perfluorocarbon ultrasound contrast agent comprising microbubbles containing a filmogenic protein and a saccharide
US5567415A (en) Ultrasound contrast agents and methods for their manufacture and use
DE69526491T3 (en) PROCESS FOR PRODUCING HOLY MICROSPHAKERS
US6080386A (en) Suspended ultra-sound induced microbubble cavitation imaging
US6068600A (en) Use of hollow microcapsules
WO1999061058A1 (en) Method of altering heartbeat
US6439236B1 (en) Methods for inducing atrial and ventricular rhythms using ultrasound and microbubbles
US8586005B2 (en) Ultrasound contrast agent dosage formulation
US6264918B1 (en) Hollow microcapsules for methods of ultrasound imaging
IL179594A (en) Ultrasound contrast agent dosage formulation
WO1999017810A1 (en) Methods of ultrasound imaging using echogenically persistent contrast agents
JP2011140527A (en) Dosage formulation of ultrasound contrast agent
Dolan et al. Increasing the dose and rate of Albunex infusion leads to superior left ventricular contrast effect
Wible Jr et al. Effects of transducer frequency and output power on the ultrasonographic contrast produced by Optison using fundamental and harmonic imaging techniques.
EP1609483B1 (en) Ultrasound contrast agent dosage formulation
Sonne et al. Left ventricular opacification after intravenous injection of Albunex®: The effect of different administration procedures
MELTZER 8. Clinical uses of contrast agents--practical considerations JANINE R. SHAPIRO, SHIMON A. REISNER
Shapiro et al. Clinical uses of contrast agents—practical considerations
Venketasubramanian et al. Stroke patterns and ipsilateral carotid stenosis among ischemic stroke patients in Singapore
MXPA96002851A (en) Preparation of microcapsules hue
KR20070039027A (en) Ultrasound contrast agent dosage formulation
NZ551986A (en) Ultrasound contrast agent dosage formulation
ZA200609760B (en) Ultrasound contrast agent dosage formulation

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW SD SL SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
NENP Non-entry into the national phase

Ref country code: KR

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
WWE Wipo information: entry into national phase

Ref document number: 09762761

Country of ref document: US